Hyphosphere microorganisms facilitate hyphal spreading and root colonization of plant symbiotic fungus in ammonium-enriched soil

Abstract

Anthropogenic nitrogen inputs lead to a high ammonium (NH₄⁺)/nitrate (NO₃^-) ratio in the soil, which restricts hyphal spreading of soil fungi. Access of symbiotic fungi to roots is a prerequisite for plant-fungal interactions. Hyphosphere bacteria protect fungi from environmental stress, yet the impact of hyphosphere bacteria on adaptation of host fungi to NH₄⁺-enriched conditions remains unclear. By developing soil microcosm assays, we report that a plant-symbiotic fungus, Phomopsis liquidambaris, harbors specific hyphosphere bacteria that facilitate hyphal spreading and assist in the root colonization in NH₄⁺-enriched soil. Genetic manipulation, 16S rRNA gene analysis and coinoculation assays revealed that the genus Enterobacter was enriched in the hyphosphere of NH₄⁺-sensitive wild-type compared to NH₄⁺-preferring nitrite reductase-deficient strain. The representative Enterobacter sp. SZ2-promoted hyphal spreading is only evident in nonsterilized soil. We further identified an increased abundance and diversity of ammonia-oxidizing archaea (AOA) and a synchronously decreased NH₄⁺:NO₃^- ratio following SZ2 inoculation. Microbial supplementation and inhibitor assays showed that AOA-mediated reduction in NH₄⁺:NO₃^- ratio is responsible for SZ2-enhanced fungal adaptation to NH₄⁺-enriched conditions. The Ph. liquidambaris-Enterobacter-AOA triple interaction promoted rice growth in NH₄⁺-enriched soil. Our study reveals the essential role of hyphosphere microorganism-based hyphal spreading in plant-fungal symbiosis establishment within nitrogen-affected agroecosystems.

Fig. 1. Soil microorganisms assist Ph. liquidambaris in spreading to the rhizosphere of rice and root colonization under NH₄⁺-enriched conditions.

A Ph. liquidambaris biomass in the soil conditions. Mycelia of Ph. liquidambaris were prepared from potato dextrose broth (PDA) medium. Ph. liquidambaris plugs were inoculated to the root-free compartment of the microcosm with sterilized soil or nonsterilized soil. The root and root-free compartments were separated by a sterile 30 μm mesh. Roots were confined to the root compartments, whereas Ph. liquidambaris in the root-free compartment were able to cross the mesh and enter root compartment. For pre-inoculated treatment, Ph. liquidambaris was preinoculated for 3 days with the mycelia kept in the 0.45 μm mesh belt to avoid mycelium diffusing into the soil. After 7 days of incubation, the Ph. liquidambaris biomass was quantified by quantitative polymerase chain reaction (qPCR) using specific internal transcribed spacer (ITS) primers. Values represent the means ± SD (n = 3). Different lowercase letters indicate significant differences between treatments (analysis of variance (ANOVA) analysis, Tukey’s HSD test with Bonferroni correction, p < 0.05). B Fluorescence microscopy showed the spread of hyphae of Ph. liquidambaris to a distance of 25–30 mm in the nonsterilized soil. C, D Fluorescence microscopy showed the attachment and colonization of hyphae on rice root surface when the inoculation site was preinoculated with hyphal mesh belt. Bars: B and C, 50 μm; D, 20 μm. IS inoculation site, n.s. no significance.

Fig. 2. The Enterobacter bacteria are enriched in NH₄⁺-sensitive WT hyphosphere but depleted in the NH₄⁺-preferring Δnir hyphosphere under NH₄⁺-enriched conditions.

A A Principal Coordinates Analysis (PCoA) is plotted based on the Bray–Curtis distance metrices for bacterial amplicon sequence variants (ASVs). PERMANOVA (Adonis function, 999 permutations) was used to test differences among treatments (n = 5). B Bacterial richness (S_obs) and diversity (Shannon index). Values represent the means ± SD (n = 5). Different lowercase letters indicate significant differences between treatments (analysis of variance (ANOVA) analysis, Tukey’s HSD test with Bonferroni correction, p < 0.05). C, D A comparison of the WT hyphosphere-enriched bacterial taxa indicates that WT preferentially attract Enterobacter in the hyphosphere. Heatmaps of the bacteria family (C) and genus (D) whose relative abundance was significantly different between treatments (Student’s t test, *p < 0.05, **p < 0.01, ***p < 0.001). A generalized logarithm transformation was applied to the fold change of relative abundance between treatments, the bacterial taxa with significant difference between WT and Δnir hyphosphere were shown in the left column and arranged in the order of log10(fold change). E Relative abundance of representative Enterobacter ASV1558 across treatments. Values represent the means ± SD (n = 5). Asterisks indicate significant differences (Student’s t-test, *p < 0.05, and **p < 0.01). F, G Evaluation of potential interactive Enterobacter isolates with WT. F Chemotactic response of Enterobacter isolates toward WT exudates. Values represent the means ± SD (n = 5). Numbers on top of each bar indicate the relative chemotactic indexes (RCI), RCI > = 2 (marked in red) indicates strong chemotactic response. G Growth response of Enterobacter isolates toward WT exudates. Enterobacter isolates were cultured with (E+)/without (E-) Ph. liquidambaris exudates for 12 h. The data were expressed as the ratio of OD₆₀₀ between E+ and E- treatments. Values represent the means ± SD (n = 6). Different lowercase letters indicate significant differences between treatments (analysis of variance (ANOVA) analysis, Tukey’s HSD test with Bonferroni correction, p < 0.05). WT wild-type, Δnir mutant strain, NS natural soil.

Fig. 3. Analysis of Ph. liquidambaris-Enterobacter sp. SZ2 interaction.

A Abundance of Enterobacter sp. SZ2 in the soil with or without inoculation of WT or Δnir under NH₄⁺-enriched conditions. Values represent the means ± SD (n = 6). Different lowercase letters indicate significant differences between treatments (analysis of variance (ANOVA) analysis, Tukey’s HSD test with Bonferroni correction, p < 0.05). B Effects of exudates secreted from WT or Δnir on SZ2 growth. Values represent the means ± SD (n = 4). Different lowercase letters indicate significant differences between treatments (analysis of variance (ANOVA) analysis, Tukey’s HSD test with Bonferroni correction, p < 0.05). Effects of exudates secreted from WT mycelia on the biofilm formation (C), swimming (D) and swarming (E) motilities. Values represent the means ± SD (n = 5). Asterisks indicate significant differences between treatments (Student’s t test, **p < 0.01, and ***p < 0.001). F–K SEM images showing SZ2 was recruited to the WT hyphosphere zone in the soil under NH₄⁺-enriched conditions (see Fig. S9). F No fungal network without Ph. liquidambaris inoculation. Fungal networks on the soil surface (G) and in soil pores (H) at 3 days post inoculation (DPI). I, J Enterobacter sp. SZ2 attached to and dispersed from mycelial networks of Ph. liquidambaris hyphal at 5 DPI (white arrows). K Enterobacter sp. SZ2 enriched in WT hyphosphere soil (white circle) at 7 DPI. WT wild-type, Δnir mutant strain, NS natural soil, SEM scanning electron microscope. Bars: F 50 μm; G, H 20 μm; I 10 μm; J, K 5 μm. WT wild-type; Δnir mutant strain.

Fig. 4. Enterobacter sp. SZ2 enhances Ph. liquidambaris adaption to NH₄⁺-enriched condition by reducing hyphosphere NH₄⁺:NO₃⁻ ratio.

A NH₄⁺/NO₃⁻ ratio in the natural soil, WT and Δnir hyphosphere soil. Values represent the means ± SD (n = 5). Different lowercase letters indicate significant differences between treatments (analysis of variance (ANOVA) analysis, Tukey’s HSD test with Bonferroni correction, p < 0.05). B Correlation relationship between relative abundance of Enterobacter (value + 1) and relative NH₄⁺/NO₃^- ratio in NS, WT and Δnir treatments. C Colony diameter of WT Ph. liquidambaris cultured on MSM was reduced as the NH₄⁺/NO₃⁻ ratio increased (soil N level, Fig. S3). Values represent the means ± SD (n = 5). D Colony diameter of Enterobacter sp. SZ2 was reduced as the NH₄⁺/NO₃⁻ ratio increased. The experiments were carried out with three individual replicates, and representative micrographs are shown. E Effects of different NH₄⁺/NO₃⁻ ratios on the abundance of Enterobacter sp. SZ2 in the hyphosphere and bulk soil, and the relative ratio between hyphosphere and bulk soil. Values represent the means ± SD (n = 3). Different lowercase letters indicate significant differences between treatments (analysis of variance (ANOVA) analysis, Tukey’s HSD test with Bonferroni correction, p < 0.05). F The effects of the presence of soil microorganisms on Enterobacter sp. SZ2-regulated fungal adaption to NH₄⁺-enriched condition. Enterobacter sp. SZ2 was first inoculated in the sterilized or nonsterilized soil with high NH₄⁺:NO₃⁻ ratio for 7 days. Ph^. liquidambaris was then cultured on the soil extraction agar (SEA) for another 7 days. Values represent the means ± SD (n = 4). Asterisks indicate significant differences between treatments (Student’s t test, **p < 0.01).

Fig. 5. Ammonia oxidizing archaea (AOA) are attracted by Enterobacter sp. SZ2 and synchronously involved in SZ2-reduced NH₄⁺:NO₃⁻ ratio in the hyphosphere of Ph. liquidambaris.

A Abundance of AOA in the natural soil, WT and Δnir hyphosphere soil. Values represent the means ± SD (n = 5). Different lowercase letters indicate significant differences between treatments (analysis of variance (ANOVA) analysis, Tukey’s HSD test with Bonferroni correction, p < 0.05). Correlation relationship between relative abundance of Enterobacter (value + 1) and AOA (B), relative abundance of AOA and relative NH₄⁺/NO₃⁻ ratio (C). D Fluorescence microscopy showing Enterobacter inhibitor carvacrol oil (CAO) repressed Ph. liquidambaris (green)-SZ2 (red) interaction. Bars: 50 μm. Effects of B3 and/or SZ2 inoculation and CAO, ammonia-oxidizing inhibitors allylthiourea (ATU) or nitrapyrin (NRP) application on NH₄⁺/NO₃⁻ ratio (E) and fungal growth on the soil extraction agar (SEA) (F). Values represent the means ± SD (n = 4). Different lowercase letters indicate significant differences between treatments (analysis of variance (ANOVA) analysis with Tukey’s HSD test, p < 0.05). G The abundance of AOA among treatments. Values represent the means ± SD (n = 3). Different lowercase letters indicate significant differences between treatments (analysis of variance (ANOVA) analysis, Tukey’s HSD test with Bonferroni correction, p < 0.05). H A Principal Coordinates Analysis (PCoA) of the AOA community is plotted based on the Bray–Curtis distance metrices for taxonomical data. PERMANOVA (Adonis function, 999 permutations) was used to test differences among treatments (n = 3). I The diversity (Shannon index) among treatments. Values represent the means ± SD (n = 3). Different lowercase letters indicate significant differences between treatments (analysis of variance (ANOVA) analysis, Tukey’s HSD test with Bonferroni correction, p < 0.05). WT wild-type; Δnir mutant strain; NS natural soil.

Fig. 6. Enterobacter bacteria establish multiple interactive relationships with saprotrophic fungi with differential responses to NH₄⁺-enriched condition.

Representative saprotrophic fungi were isolated from rice rhizosphere soil. The pathogenic fungus Fusarium proliferatum Ff-1 was previously reported to cause causing bakanae disease of rice. A Growth conditions of different fungal isolates on modified MSM with NH₄⁺/NO₃^- ratio at 5/1. These strains were classified as NH₄⁺-sensitive, Alternaria sp. RH3 and F. proliferatum Ff-1; NH₄⁺-tolerant, Aspergillus sp. RH7 and Alternaria sp. RH10; NH₄⁺-preferred, Trametes sp. RH8 based on colony phenotype. Evaluation of the interaction between fungal strains and Enterobacter sp. SZ2 by detecting hyphal exudates on bacterial performance. Effects of hyphal exudates on the growth status (B) and biofilm formation (C) of Enterobacter sp. SZ2. Values represent the means ± SD (n = 5). Asterisks indicate significant differences between fungal treatment and mock treatment (Student’s t-test, *p < 0.05, and **p < 0.01). n.s. no significance.

Fig. 7. Ph. liquidambaris-Enterobacter coinoculation increases ammonia oxidizing archaea (AOA) abundance in the rice rhizosphere and promotes plant growth.

A Chemotactic response of Enterobacter sp. SZ2. Accumulation of bacteria in the capillaries was calculated as the average from the CFUs obtained in the plates. Numbers on top of each bar indicate the relative chemotactic indexes (RCI), RCI > = 2 (marked in red) indicates strong chemotactic response. B Cocolonization of Ph. liquidambaris (green) and Enterobacter sp. SZ2 (red) on rice roots. Bar: 20 μm. C Ph. liquidambaris-Enterobacter sp. SZ2 coinoculation strongly increased AOA abundance in rice rhizosphere soil. D, E Effects of WT Ph. liquidambaris and/or Enterobacter sp. SZ2 inoculation on rice shoot length, root length and seedling fresh weight in sterilized (D) or nonsterilized (E) soil conditions. Values represent the means ± SD (n = 3-5). Different lowercase letters indicate significant differences between treatments (analysis of variance (ANOVA) analysis, Tukey’s HSD test with Bonferroni correction, p < 0.05). NSE natural soil extracts; RE root exudates; HE hyphal exudates; WT wild-type.

Fig. 8. A model illustrating the fungal-Enterobacter interaction facilitating hyphae of Ph. liquidambaris spreading to the rhizosphere of rice under NH₄⁺-enriched conditions.

A Enterobacter sp. SZ2 was enriched in the hyphosphere zone under NH₄⁺-enriched condition. B Enterobacter sp. SZ2 was able to recruit ammonia oxidizing archaea (AOA) for NH₄⁺ transformation in the hyphosphere soil. C The decreased NH₄⁺:NO₃⁻ ratio facilitates hyphal growth and spreading in the soil and facilitates root-fungal colonization. D The root-fungal-Enterobacter-AOA interaction is further established and provides benefits for rice growth in the NH₄⁺-enriched paddy soil. Graphic was created with Biorender.com and Photoshop CS6 software.