Effect of living mulches on soil nutrient levels
Soil nutrients level was found to be significantly affected by the season (Table S1), but not by the living mulches (Fig. 1), although some interactions between sampling point and treatment between selected parameters (pH, salinity and P levels) occurred (Table S1).
PCA of soil chemical parameters in relation to sampling time and living mulch treatment of an organic apple orchard.
In particular, N content resulted to increase during the vegetative season: with a steady trend in case of N-NO3, with a significantly higher peak in summer for N-NH4. Phosphorus and potassium levels were the lowest in summer. Magnesium concentration tended to increase during the season (from about 78 g/l measured in May to about 97 g/l in September), while calcium levels tended to decrease (from about 300 g/l to about 240 g/l, in May and September, respectively). Fluctuations in pH were observed during the season as well, while salinity tended to increase from spring to autumn (Table S1).
However, a significant effect of sampling time and the living mulch species was observed in case of N and K (Table S1). The living mulches lowered (about 30–50%) both N-NO3 and N-NH4 and K levels in soil, particularly in summer, compared to the control (Table S1). In addition, F. vesca was the living mulch species that significantly reduced the N-NH4 concentrations during the whole season also when compared to the other two species (Table S1). The reduction of K level occurred in different seasons: summer for A. vulgaris and autumn for F. vesca. The three living mulch species affected both pH and salinity along the growing season: all three induced a decrease of salinity in autumn, while diverse fluctuation trends were recorded for each of them in case of pH values (Table S1).
Impact of living mulches on soil bacterial metabolic activity and diversity
No overall effect of either the sampling time or living mulch species was observed on bacterial activity (AWCD), bacterial biodiversity (Index H’) or substrate richness (S) measured with Biolog EcoPlates analysis (Table 1). Nevertheless, the interaction between sampling point and treatment factors were significant for AWCD and richness. Considering the combined effect of the two factors, significantly affected bacterial activity: A. vulgaris induced the lowest bacterial activity and substrate richness in comparison to control and the other two living mulch species in spring and summer. Bacterial activity of M. x piperita soil was lowered during summer compared to control or F. vesca treatment. However, these effects were temporary, as at the end of the vegetative season no significant differences were observed in bacterial activity and level of biodiversity between the treatments, and the control as well (Table 1).
Carbon sources metabolised by the bacterial community were consistently distinguished according to their utilization rate regardless the timepoint during the vegetative season and classified as: poorly, medium or highly metabolized (Fig. 2). These three groups of compounds included various classes of carbon source. Nevertheless, a noticeable high number of carbohydrates or carboxylic acids was observed in medium or highly metabolized group (upper colored strip on Fig. 2). No defined clusters of C sources were identified based on the time of sampling or the living mulch species, though the metabolic potential of some compounds was found to be significantly affected either by timepoint (11 substrates) or treatment (3 substrates) (Table S2). The metabolization of about 50% of the C sources (mainly carbohydrates) was increased in summer sampling point in comparison to other timepoints. Only two compounds (L-arginine and 4-hydroxybenzoic acid) were characterized by relatively high metabolic activity at the beginning of the vegetative season, while putrescine and three polymers (Tween 40, Tween 80 and α-cyclodextrin) showed higher utilization rate in autumn compared to spring sampling point. Interestingly, the bacterial community of the living mulches differentially affected the metabolization of only two amino acids (L-asparagine or L-threonine) and a carbohydrate (i-Erythriol). The bacterial soil community associated with M. x piperita showed high metabolic potential toward L-threonine than that of A. vulgaris, while the opposite effect was observed for i-Erythriol. The bacterial community associated with F. vesca showed an increased metabolisation of L-asparagine, compared to control.
Effect of living mulches in an organic apple orchard on soil microbial activity toward 6 classes of C sources (in total 31 compounds) along the growing season (three timepoints).
Impact of living mulches on soil bacterial community genetic diversity
The soil samples collected in summer provided 1 926 479 paired reads obtained from 16 S rDNA V3V4 amplicon sequencing. The number of reads per sample ranged between 114,749 and 180,294. After the quality control and reads merging, on average 44.71%±2.58% paired reads per sample were retained. Rarefying the library size to the smallest value provided enough sequencing depth for downstream analyses (Figure S1). Between 99.91 and 100% of the sequences were assigned to the Bacteria kingdom, the others to Archaea, prompting to analyse only the former reads.
Up to 36 different bacterial phyla were found in analysed dataset, although only 11 of them were present with at least 1% relative abundance (Table S3). Nine of them showed significantly changed abundance influenced by the living mulch species, which resulted in clustering the samples from each treatment together according to the phylum biodiversity level (Fig. 3). Bacterial communities of M. x piperita and A. vulgaris clustered together, while that of F. vesca was closer to the control.
Relative abundance of the most abundant phyla (>1%) across the samples collected from soil of an organic apple orchard managed with different living mulches or naturally covered (control).
Proteobacteria and Actinobacteriota were the most numerous bacterial phyla, with relative abundancies between 26.00 and 30.22% and 23.17–27.17%, respectively. Members of Proteobacteria were significantly more abundant in the control than in the community of any living mulch species, especially A. vulgaris. Actinobacteriota community was the highest in soil with A. vulgaris mulch (27.17%), while it was the lowest in F. vesca mulch (23.17%). Other two abundant phyla, Acidobacteriota (11.23–11.69%) and Verrucomicrobiota (5.98–6.31%) were not affected by the living mulch species. A statistically comparable relative abundance was observed for Bacteroidota either in M. x piperita and A. vulgaris mulches (8.26% or 7.51%, respectively) or F. vesca and control (11.52% or 11.77%, respectively). An opposite trend characterized the abundance of the Firmicutes phylum, which showed higher values for M. x piperita and A. vulgaris mulches (3.76–4.04%) compared to control and F. vesca (1.89–2.34%). Interestingly, four less abundant phyla (Chloroflexi, Gemmatimonadota, Myxococcota and Patescibacteria) were increased by all living mulch species compared to control. Planctomycetota was the only phylum, whose abundance was specifically enhanced by F. vesca mulch.
At the genus level, 828 different taxa were identified. Among them, 287 taxa showed the same prevalence (always absent or present) in all three samples replicates collected from each living mulch (Fig. 4). Only few genera were unique (present in all samples) to each living mulch species: four to F. vesca (Erwinia, Aquabacterium, Zixibacteria and uncultured genus representing Actinomarinales order), two to A. vulgaris (Afipia and Azotobacter), one to M. x piperita (Microbacteriaceae) and three present only in control treatment (Planctmicrobium, Janthinobacterium, unclassified genus representing Verrucomicrobiaceae family). Eight taxa were found in all living mulches, and not in the control, but their relative abundance did not exceed 0.1%. These included: OLB12, unclassified genus from Gemmatimonadaceae family, Candidatus Magasanikbacteria, Candidatus Woesebacteria, Candidatus Yanofskybacteria, S-BQ2-57 soil group, Subgroup 15 and HSB OF53-F07.
Venn diagram based on the counts of bacterial taxa with uniform prevalence (totally absent or totally present within individual treatment) in the soil samples from an organic apple orchard managed with different living mulches. The taxa specific to each living mulch and the core taxa identified in all treatments excluding control are listed in the boxes.
As many as 213 genera showed significant differences between the living mulches, with only 11 taxa not showing changes of relative abundance in any living mulch species when compared to control: Luteolibacter, Gaiella, Pirellula, Aetherobacter, Nitrospira, Pedosphaeraceae, Dyadobacter and uncultured or unclassified genera representing Caulobacteraceae, Steroidobacteraceae families or Chitinophagales order. Ten genera were differently affected by the living mulch species, but not between them and control treatment. The changes in abundance compared to control were positive for 106 genera and negative for 91 genera (Fig. 5a). Mixed response (increased and decreased abundance depending on the living mulch species) was observed only for 8 genera: Skermanella, NB1-j, Dongia, Geogfuchsia, Chujaibacter, Puia, OM190 and unclassified genus representing Intrasporangiaceae family.
Venn diagrams based on the number of bacterial taxa with uniform prevalence (totally absent or totally present within individual treatment), which showed significant positive or negative changes in abundance in comparison to the control. a – overall number of taxa with significantly changed abundance, b – taxa with significantly increased abundance with different living mulch species, c – taxa with significantly decreased abundance with different living mulch species.
The abundance of several groups of genera was either increased or decreased by a single or two or all living mulches (Fig. 5b and c, details in Table S4). Positive changes shared by all living mulches interested 23.7% of taxa, while 33.3% were shared by two mulching species (Fig. 5b). About 57.6% negative variations were shared by the three living mulches and 13.1% by two species (Fig. 5c). The positive changes unique to each mulching species occurred to about 11.4–12.3% of taxa in case of M x piperita and F. vesca, and to 19.3% in case of A. vulgaris. For the latter species, a similar order of magnitude was observed for negative changes (20.2%), while these affected only about 4.0–5.1% of taxa in case of F. vesca and M. x piperita, showing a much more homogeneous response of the bacterial communities of all three living mulches.
F. vesca showed the lowest total number of genera with increased (55) or decreased (67) relative abundance in comparison with the other two species. The most noticeable changes at genus level between mulches and control were the decreased abundance of: Flavobacterium, Pseudomonas, Subgroup 10, Thermomonas and Amaricoccus, paralleled by increased abundance of Microscillaceae, Elsterales, Subgroup 2, Acidobacteriales and Candidatus Solibacter. (Table S4).
In an effort of verifying the trends induced by the living mulches on the bacterial community, a PCA analysis of soil bacterial diversity data of the current study and of a previous study (data from Furmanczyk et al.10) was performed (Fig. 6). The analysis pointed out that, after four years of establishment, the bacterial populations of the living mulches were similar to that of the control (natural cover). The modifications induced by the establishment of the mulches (i.e. at year zero) or after their first growing season were much higher. Interestingly, the PCA pointed to a potential different path for the bacterial population of the living mulches to reach the “equilibrium” of the natural cover (control). Notwithstanding the similarity of the bacterial communities in the medium-term, a significant increase in biodiversity (expressed by both Shannon and Simpson indexes) was observed for soils mulched with F. vesca and M. x piperita, compared to control (Table 2). Although not statistically confirmed, such trend was also observed for the community richness (Chao1 index).
PCA of soil bacterial relative abundance at genus level in relation to sampling point and living mulch. The arrows show the direction of changes between specific treatments according to the sampling year.
Correlations between soil nutrient content, bacteria activity and biodiversity
To evaluate the impact of the mulching species on the relations between soil chemical properties and the bacterial activity and biodiversity, a correlation analysis with data obtained from the summer sampling point was performed. To increase the power of the analysis, the sequencing data of only 20 positively and 20 negatively affected taxa for each living mulching species were considered, leaving out non-culturable taxa known only from sequencing environmental samples and taxa identified as “candidatus”. To further increase the quality of potential correlations, data from the previous study carried out on the same trial10 were also added to the dataset.
The most significant correlations, out of more than 750 significant ones (Table S5), between soil chemical and biological data and taxa are presented on Fig. 7. P and K content showed the highest number of correlations with bacterial taxa (17 and 12 cases, respectively) Metabolism of several compounds (7 out of 14), included in the category of medium metabolized compounds, was correlated with relative abundance of 25–35% of the bacterial genera. These compounds included carbohydrates and polymers (β-methyl-D-glucoside, D-galactonic acid γ-lactone, α-D-lactose, D-xylose, glycogen and α-cyclodextrin). Only three C sources (D-mannitol, L-serine, L-arginine) from highly metabolized group and i-erythritol from poorly metabolized compounds resulted correlated with 20–24% of the bacterial taxa. Ten genera (Luteitalea, Dongia, Jatrophihabitans, Marmoricola. Nitrospira, Acidibacter, Bradyrhizobium, Reyranella, Variovorax and Luteolibacter) showed no correlation with any soil chemical property or C sources. Significant correlations with 20–25% of the analysed bacterial taxa emerged for 11 genera (Pseudomonas, Amaricoccus, Buttiauxella, Paenibacillus, Sphingopyxis, Cellulomonas, Ilumatobacter, Nakamurella, Pseudoarthrobacter, Skermanella and Sphingomonas). Interestingly also AWCD and Shannon Index were correlated with nearly a third of the analysed microbial genera (Table S5).
Heatmap showing the significant correlations (p < 0.05) between selected soil bacteria taxa (excluding Candidatus genera and the taxa not assigned to genus level) and soil chemical parameters and C sources utilised to assess microbial activity. Data of samples gathered in summer from soil managed with living mulch species or natural cover.
