OJ and LP seedling growth and plant traits
OJ and LP seedlings were exposed to shade stress and examined to determine their growth response to this stress (Fig. 1, Additional file 1: Table S1). Shade treatment resulted in different growth suppression in the two plants. Shade did not significantly influence leaf area in OJ but resulted in a 14.9% decrease (P < 0.05) in LP leaf area when leaves were exposed to 14 d of shade stress compared to non-shade (Fig. 1A). Shade treatment significantly decreased (P < 0.01) total root length, root surface area, and root volume in LP, while OJ exhibited superior acclimation to shade stress (Fig. 1B-D). In addition, OJ and LP had different changes in chlorophyll content in response to shade stress. Shade stress increased chlorophyll content in OJ, while shade stress reduced chlorophyll content in LP (Fig. 1E). Fluorescence parameters (Fv/Fm) for chlorophyll a were reduced significantly in LP compared with OJ, indicating that OJ maintained higher photosynthetic capacity under shade stress (Fig. 1F). These results demonstrate that OJ is more shade-tolerant than LP.
Shade-intolerant LP was more responsive than shade-tolerant OJ to shade and soil microbes (Fig. 2; Additional file 2: Table S2). Shade stress negatively affected total LP plant biomass during the experimental period, but no significant change was observed in OJ (Fig. 2A, B). The total dry biomass and shoot biomass in LP were lower in live soil compared to those in sterile soil. This occurred both under non-shade and shade stress. In contrast, no significant difference was observed in OJ under the same conditions.
For LP, root: shoot ratios were higher in the shade compared to non-shade conditions. LP also exhibited higher ratios in sterile compared to live soil under shade stress, however there was no significant difference in light response between live and sterile soil (Fig. 2C). Shade significantly increased (P < 0.05) LP specific root length, but we observed no significant change between sterile and live soil. We did observe a slight difference of specific root length in OJ under light and soil treatments (Fig. 2D).
As observed with LP specific root length, LP specific leaf area only responded to shade treatment, with higher values (P < 0.05) in shade compared to non-shade (Fig. 2E). Plant leaf and root N content showed significant responses (P < 0.05) to shade treatment. The synergistic effect of shade treatment and the presence of soil microbes on root and leaf N was not significant (Fig. 2F, G; Additional file 2: Table S2). Compared to non-shade treatment, shaded plants in sterile soil had a 17.18% decrease in leaf N content and shaded plants in live soil displayed a 36.52% decrease in leaf N content. LP root N content decreased 18.73% and 24.57% in shaded versus non-shaded plants under sterile and live soil treatments, respectively. For OJ, shade increased the N content in live soil: 30.61% in leaves and 33.01% in roots. The corresponding values for shaded sterile soil were 26.58% and 28.45% for leaves and roots, respectively (Fig. 2F, G).
Soil treatment and species interaction significantly affected plant shade response index (Fig. 3A; Additional file 3: Table S3). In LP, the plant shade response index responded to soil treatments with significantly higher values (P < 0.05) in live soil versus sterile soil, while there was no difference in OJ (Fig. 3A). Also, shade and species interaction had a significant effect on soil-plant feedback index (Fig. 3B; Additional file 3: Table S3). Soil-plant feedback index in LP was consistently lower (P < 0.05) in shade compared to non-shade treatments, but there was no difference in the value of OJ between highlight and shade treatments (Fig. 3B).
Soil chemical characteristics
Shade stress significantly influenced most of the physicochemical properties analyzed (Table 1). Both OJ and LP soil showed significant increases in the NO3−-N content with shade treatment (P < 0.001). Conversely, shade treatment decreased TP, TK and AK in both soil types compared to non-shade treatment, with a greater effect with LP soil. Shade treatment of OJ resulted in a significant decrease in rhizosphere NH4+-N and a significant increase in the rhizosphere AP content. The opposite trend was observed with LP. Shade treatment had a small effect on the soil TN, SOC, C:N ratio, and rhizosphere pH level.
Bacterial diversity and community composition response to shade stress
Amplicon products of the V4 region of the 16S rRNA gene were obtained from each of the 60 samples and sequenced using the Illumina HiSeq 2500 platform. A total of 5,371,314 bacterial clean reads were obtained. These sequences were grouped into 11,485 OTUs according to a 97% similarity threshold. According to the rarefaction curves (Additional file 4: Figure S1), the sequencing depth in these samples was sufficient to cover the full diversity.
The OJ and LP rhizosphere soil bacterial communities did not have similar alpha diversity features, as measured by the OTU richness, Shannon’s diversity index (H) and Simpson’s Evenness (E) (Fig. 4). The OTU richness and diversity did not show significant differences between the two rhizosphere soils. However, the evenness increased (P < 0.05) in OJ soil under shade stress but decreased in LP soil. This suggests that a few numerically dominant OTUs inhabit the LP rhizosphere.
The patterns of bacterial community composition between treatments in OJ and LP soils were analyzed using PCoA based on Bray-Curtis dissimilarity. The PCoA analysis explained 64.06% of variation (two axes) in bacterial community composition. Shade treatments led to a distinct bacterial community structure (PERMANOVA, P < 0.05), and the bacterial community structures between the OJ and LP rhizosphere soils were also obviously different (Fig. 5). Further evidence showed that the bacterial communities collected within the OJ rhizosphere on the one hand, and JP rhizosphere on the other, overlapped partially in the PCA plot (Additional file 5: Figure S2), indicating that OJ and LP soils had different bacterial community structures.
In both OJ and LP rhizospheric soil, the edaphic bacterial communities harbored 11 different phyla (accounting for more than 93% in each sample). The most numerically dominant phyla were Proteobacteria followed by Acidobacteria and Thaumarchaeota (Fig. 6A). Proteobacteria, Actinobacteria, and Chloroflexi decreased in LP soil in response to shade stress, but an increase or a lower degree of change was observed in OJ soil. In contrast, shade led to higher abundances of Verrucomicrobia and Acidobacteria in LP soil, compared to OJ soil (Kruskal-Wallis, P < 0.01).
There were 12 genera (> 0.5%) within the classes Alpha and Gamma Proteobacteria, Flavobacteria, Planctomycetia, Spartobacteria, Nitrospira, and Thaumarchaeota. The genus Candidatus Nitrososphaera was clearly dominant within the taxonomic structure of the bacterial community (Fig. 6B). The most evident differences between OJ and LP rhizosphere soil bacterial communities were the opposing trends in the abundance of Nitrospira, Steroidobacter, Kaistobacter and Pirellula. These genera were unchanged or increased with increasing shade treatment in OJ soil, but they tended to decrease in LP soil. In contrast, the relative abundance of Rhodoplanes, Planctomyces, and Pseudomonas was larger (Kruskal-Wallis, P < 0.01 or P< 0.001) in OJ soil under shade treatment, compared to LP soil. In LP soil Gemmata was more abundant than in OJ soil (Kruskal-Wallis, P< 0.001), although shade stress decreased the relative abundance of this genus in both soils.
Core microbial players associated with rhizosphere soil in OJ and LP
The core bacteriome of OJ and LP rhizosphere soils was determined to examine shifts in the bacterial communities observed with the different host types. This analysis suggested that a specific taxonomy may exist which is particularly well adapted and prominent under different growth conditions. We found that OJ rhizosphere soil was dominated by OTUs identified as: Nitrosovibrio (19.1% of total core bacterial OTU), Aquicella (12.5%), Planctomyces (11.8%), Pseudomonas (11.2%), Nitrospira (10.3%), Steroidobacter (10.3%), Flavobacterium (8.8%), Kaistobacter (5.2%), Bacillus (6.8%), and Rhodoplanes (5.1%), which mostly belongs to Proteobacteria. In contrast, the LP rhizosphere soil was dominated by Acinetobacter (21.0%), Flavisolibacte (19.3%),and Skermanella (17.1%) (belonging to Proteobacteria and Bacteroidetes, respectively). Nitroso vibrio tenuis and Candidates Nitrososphaera_SCA1145 (both 5.9%) were identified in theOJ rhizosphere soil core (Additional file 6: Table S4).
Relationships between shade-tolerant parameters and bacterial communities
There was a significant positive relationship between plant shade tolerance and soil bacterial community composition (Fig. 7). Among all the shade-tolerant indicators measured, leaf area, Fv/Fm, chlorophyll content, and root morphology were correlated with soil bacterial community composition (P < 0.001 for all).
Relationships between bacterial community and environmental variables
The OJ and LP soil bacterial community structures displayed clear, individual correlations (P < 0.001 or P < 0.05) to soil physicochemical variables including NH4+-N, NO3−-N, and TK as shown by the Mantel test (Additional file 7: Table S5). CCA analysis revealed that the OJ and LP rhizosphere soil bacterial communities were affected differently by edaphic chemical parameters under the shade treatments examined. The proportion of total variability of OJ and LP soil bacterial communities attributed to the explanatory variables was 73.21% and 82.57%, respectively. This partition of variability was significant (general permutation test, P < 0.01 or 0.05; 999 replicates; Fig. 8; Additional file 8: Table S6). AK and total N were the major factors affecting the bacterial assemblages in OJ soil as judged by the length of the vectors shown in our CCA plots. In OJ soil, AK and total N were positively correlated (P < 0.05) with Gemmatimonadetes, Chloroflexi, Acidobacteria, Nitrospirae, and WS3. For OJ soils, CCA was consistent with the trends revealed by PCA showing a clear separation between control and shade treatment (Additional file 5: Figure S2). The TN, NO3−-N, and NH4+-N concentrations, three directly interlinked parameters, had a strong effect on bacterial assemblages in the LP soil. TN and NH4+-N were positively correlated (P < 0.05) with Actinobacteria, Bacteroidetes, and Thaumarchaeota. Taxa, such as Verrucomicrobia, Chloroflexi, Acidobacteria, Planctomycetes, Gemmatimonadetes, and WS3 were positively correlated (P < 0.01) with NO3−-N. Additionally, shade treatments of different durations separately clustered in LP soil.