Effects of shade stress on morphophysiology and rhizosphere soil bacterial communities of two contrasting shade-tolerant turfgrasses

Background: Perturbations in the abiotic stress directly or indirectly affect plants and root-associated microbial communities. Shade stress presents one of the major abiotic for growth, as light availability is severely reduced under a leaf canopy. Studies have shown that shade stress influences plant growth and alters plant metabolism, yet little is known about how it affects the structure of rhizosphere soil bacterial communities. In this study, a glasshouse experiment was conducted to examine the impact of shade stress on the physiology of two contrasting shade-tolerant turfgrasses and their rhizosphere soil microbes. Shade-tolerant dwarf lilyturf (Ophiopogon japonicus, OJ) and shade-intolerant perennial turf-type ryegrasss (Lolium perenne, LP) were used. Bacterial community composition was assayed using high-throughput sequencing. Results: Our physiochemical data showed that under shade stress, OJ maintained higher photosynthetic capacity and root growth, thus OJ was found to be more shade-tolerant than LP. Illumina sequencing data revealed that shade stress had little impact on the diversity of the OJ and LP’s bacterial communities, but instead impacted the composition of bacterial communities. The bacterial communities were mostly composed of Proteobacteria and Acidobacteria in OJ soil. Further pairwise fitting analysis showed that a positive correlation of shade-tolerance in two turfgrasses and their bacterial community compositions. Several soil properties (NO3--N, NH4+-N, AK) showed a tight coupling with several major bacterial communities under shade stress, indicating that they are important drivers determining bacterial community structures. Moreover, OJ shared core bacterial taxa known to promote plant growth and confer tolerance to shade stress, which suggests common principles underpinning OJ-microbe interactions. Conclusion: OJ was more shade-tolerant than LP. Shifts in rhizosphere soil bacterial community structure play a vital role in shade-tolerance of OJ plants. of these associated microbes, our work using two turfgrass genotypes with contrasting shade tolerance profiles provides a description of the physiological and rhizosphere soil bacterial response induced by shade stress. We uncovered several differences between OJ and LP through analysis of the physiological response and growth suppression that accompanied shade stress. Our physiochemical data demonstrated that shade stress resulted in more severe growth suppression in LP than in OJ. This was indicated by a larger decline in leaf area, total root length, root volume, and surface area in LP versus OJ. Similar results have been observed in several tree species, showing that shade-tolerant red oak had greater leaf area and dry mass than shade-intolerant species [11]. Plant photosystem II is sensitive to various environmental stresses, including shade stresses Chlorophyll a fluorescence ( F v / F m ) is a valuable indicator of stress tolerance 13]. Our results show that under shade stress OJ maintained higher F v / F m and chlorophyll ( a + b ) content, suggesting it has a better photosynthetic capacity under shade stress. The present data agrees with our first hypothesis, listed above. These findings suggest that OJ is more shade-tolerant than LP. community Maintenance photosynthetic capacity growth during shade stress shade-tolerant than LP. Moreover, shifts in rhizosphere soil bacterial community structure play a vital role in shade-tolerance of OJ and LP plants. The also shows that, under shade stress, some soil properties showed a tight coupling with several major bacterial communities, indicating that they are important drivers determining bacterial community structures. correlations between soil physicochemical bacterial between

grass growth. It has been estimated that approximately 20-25% of all grassed areas in the USA [1], and 50% of turfgrass in China, is subjected to varying degrees of shade [2]. Thus, insight into the mechanism of turf grass resistance or adaptation to shade stress is vital for turf management and selection of shade-tolerant turf grass varieties.
The negative effects of shade on plant morphology and physiology have long been established. The morphology of shaded leaves is characterized by an increase in specific leaf area and a decrease in thickness [3,4]. Particularly, a lack of light adversely impacts chlorophyll content, chloroplast ultrastructure, as well as photosynthetic physiological processes [5,6].
Root-associated bacterial communities play a crucial role in maintaining the health of the plant host [7]. These communities possess complex relationships where the composition and abundance of microbial communities depends on factors such as: soil chemical properties, plant genotype and phenotype, and perturbations in the surrounding abiotic or biotic stresses [8,9]. The root distribution pattern in soil reflects plant ecological adaptation and may increase plant survival under stress [10].
However, there is little understanding how shade influences root morphology and root-associated bacterial communities. Clarifying exactly how shade stress affects soil bacterial communities is an essential step in developing strategies to combat shade in turfgrass management.

Results
Plant growth characteristics in response to shade stress OJ and LP seedlings were exposed to shade stress and examined to determine their growth response to this stress (Fig. 1). 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, Additional file 1: Table S1). 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 (F v /F m ) 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.

Soil chemical characteristics
Shade stress significantly influenced most of the physicochemical properties analyzed (Table 1). Both OJ and LP soil significantly increased the NO 3 − -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, however there was a greater effect with LP soil. Shade treatment of OJ resulted in a significant decrease in rhizosphere NH 4 + -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 5371314 bacterial clean reads were obtained. These sequences were grouped into 11485 OTUs according to a 97% similarity threshold.
According to the rarefaction curves (Additional file 2: Figure S1), the sequencing depth in these samples was sufficient to cover the full diversity.
The bacterial communities did not have similar alpha diversity features between OJ and LP rhizosphere soil, as measured by the OTU richness, Shannon's diversity index (H) and Simpson's Evenness (E) (Fig. 2). The richness and diversity of OTUs 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. 3). Further evidence showed that the bacterial communities collected at OJ rhizosphere on the one hand, and JP rhizosphere on the other, overlapped partially in the PCA plot (Additional file 3: 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 principally 11 different phyla (accounting for more than 93% in each sample). The most numerically dominant phyla were Proteobacteria followed by Acidobacteria and Thaumarchaeota (Fig. 4A). 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. In all the samples, the taxonomic structure of the bacterial community was characterized by a clear numeric dominancy of the genus Candidatus_Nitrososphaera (Fig. 4B). The most evident differences between OJ and LP rhizosphere soil bacterial communities were the opposing trends in the abundance of Nitrospira, Steroidobacter,  Table S2).

Relationships between shade-tolerant parameters and bacterial communities
There was a significant positive relationship between plant shade tolerance in OJ and LP plants and soil bacterial community composition (Fig. 5). Among all the shade-tolerant indicators measured, leaf area, F v /F m , chlorophyll content, and root morphology were significantly 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 NH 4 + -N, NO 3 − -N, and TK as shown by the Mantel test (Additional file 5: Table S3). CCA analysis revealed that the OJ and LP rhizosphere soil bacterial communities were affected differently by edaphic chemical parameters under the different 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. 6; Additional file 6: Table S4). 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 3: Figure S2). The TN, NO 3 − -N, and NH 4 + -N concentration, three directly interlinked parameters, had a strong effect on bacterial assemblages in the LP soil. TN and NH 4 + -N were positively correlated (P < 0.05) with Actinobacteria, Bacteroidetes, and Thaumarchaeota, respectively. Taxa, such as Verrucomicrobia, Chloroflexi, Acidobacteria, Planctomycetes, Gemmatimonadetes, and WS3 were positively correlated (P < 0.01) with NO 3 − -N.
Additionally, shade treatments of different durations separately clustered in LP soil.

Discussion
The current knowledge of the plant shade stress response has arisen from studies of physiology and morphology and has neglected the function of root-associated soil microorganisms. Because plant health is closely tied to the activity of these associated microbes, our work using two turfgrass genotypes with contrasting shade tolerance profiles provides a description of the physiological and rhizosphere soil bacterial response induced by shade stress. Plant shade tolerance is related to bacterial community composition Plants and their root-associated microbial communities are strongly interlinked, as a result, perturbations in the abiotic stress directly or indirectly influence plants, their associated microbial communities as well as the interaction between these organisms [8] . In both OJ and LP, we found that shade stress had little impact on bacterial richness and soil community diversity, which is consistent with other studies showing that community diversity is not significantly impacted by drought [14,15].
Similar results have been reported in salt stress demonstrating that increasing salinity has no effect on total bacterial community richness [16]. The observed shifts in the soil microbiome when OJ and LP were shade stressed involved changes in relative bacterial abundance, rather than outright abolition of shade susceptible taxa and concomitant appearance of tolerant ones. This helps explain the lack of change in alpha-diversity.
Bacterial community composition was significantly different between OJ and LP rhizosphere soils with various shade treatments. The relative abundance of Proteobacteria and Actinobacteria have been shown to accumulate in OJ soil in response to shade stress, while they have been shown to decrease in LP soil. Accumulating evidence shows that Proteobacteria and Actinobacteria display different trends in response to various environmental stresses, such as drought, salt and heavy metal stresses [17][18][19][20]. Actinobacteria are implicated in promoting plant growth under stress [21]. Many of them are known to form spores, which are resistant to adversity and can survive under stress conditions [22,23]. suggests that OJ preferred these genera and they may be markers of better shade-tolerance in OJ.
To further clarify this assumption and study the correlation of shade-tolerance parameters and bacterial community composition, pairwise fitting analyses were performed to compare above and below-ground morphology, photosynthetic capacity and bacterial community composition. We found that the leaf area, root volume, surface area, F v /F m , and chlorophyll (a+b) content were positively and significantly related to soil bacterial community composition. This observation is in line with our second hypothesis, and with the last part of our third hypothesis. Similar observations have also been shown in maize with differing aluminum tolerances. It was observed that maize cultivars that depended on Al tolerance altered their root morphology and rhizosphere diazotrophic community composition [28].

Changes in soil physicochemical
properties play an essential role in shaping bacterial communities under shade stress Soil acts as a strong ecological filter affecting the bacterial community structure and diversity.
Numerous studies of microbial communities under abiotic stress have shown that soil factors govern microbial community structure [29][30][31]. Bottomley et al. [32] observed that soil NH 4 + -N was a dominant environmental factor that influenced bacterial community structures. This is due to the fact that it is the main nitrogen source for bacteria as seen by 15 N isotope tracing [33]. Similarly, Nguyen et al. [34] also reported that bacterial diversity and composition were related to soil NH 4 + -N and total N content exposed to post-waterlogging or post-prolonged drought. Consistent with our third hypothesis, the major drivers in OJ rhizosphere soil were AK and total N which was positively associated with Gemmatimonadetes, Chloroflexi, Acidobacteria, Nitrospirae, and WS3. Thus we confirmed the importance of these two soil variables in regulating the OJ rhizosphere. In contrast, total N, NO 3 --N, and NH 4 + -N concentration exhibit a strong effect on bacterial assemblages in LP soil.
A strong relationship between soil physicochemical properties and bacterial communities was also observed in water-limited soils. In these soils, the abundance of Acidobacteria correlated positively with soil NH 4 + -N and total P and negatively with total N and Mg 2+ , whereas Chloroflexi displayed the opposite trend [35].
In both control and stressed soil, host species were confirmed to exert a significant influence on bacterial community structures [36]. Our PCoA data shows that bacterial communities separated between OJ and LP soil. Al-tolerant maize cultivation significantly influenced the diazotroph populations [28], a result that aligns with our results with turfgrass . This may be mainly attributed to plant root exudates, which are key determinants of microbial community composition in plantmicroorganism interactions [37].

Conclusions
This study describes the physiological plant shade stress response as well as the rhizosphere soil bacterial community shade stress response in two turfgrass genotypes with different shade tolerances . Maintenance of higher photosynthetic capacity and root growth during shade stress in OJ could make this more shade-tolerant than LP. Moreover, shifts in rhizosphere soil bacterial community structure play a vital role in shade-tolerance of OJ and LP plants. The study also shows that, under shade stress, some soil properties showed a tight coupling with several major bacterial communities, indicating that they are important drivers determining bacterial community structures.

Methods
Glasshouse experimental setup and soil sampling

Determination of plant growth characters
The total leaf area for each seedling was measured in the laboratory using a LI-3000A leaf area scanner (LI-COR Inc., USA). Root morphology including total root length, root surface area, and root volume was analyzed using a WinRhizo-V700 root scanner (Regent Instruments Inc., Quebec, Canada). The chlorophyll content was determined spectrophotometrically using 80% acetone as a solvent [38] (Lichtenthaler, 1987). On the same leaf, a portable pulse-modulated fluorometer (PAM2100, Walz, Effeltrich, Germany) with the PamWin software was used to measure chlorophyll fluorescence (F v /F m ).

Soil physicochemical analyses
Soil pH was measured using a pH meter (Mettler Toledo FE20, Switzerland) in a soil solution with a 1:2.5 soil: water ratio. The NH 4 + -N and NO 3 − -N were extracted with 2.0 M KCl and measured by a continuous flow analyzer (Flowsys, Systea Inc., Italy). Soil was processed for C content by first removing inorganic C through treatment with 1 M HCl. Following removal of inorganice C, soil organic C was analyzed using an auto-analy zer (Shimadzu, Kyoto, Japan). The total N in the soils were measured on an elemental analyzer (ECS 4024, Costech Inc., Italy). Total P was determined by digesting samples first with HClO 4 -H 2 SO 4 ,followed by the molybdenum blue method using an ultraviolet-visible spectrophotometer (UV-1000, AOE Instruments, Shanghai, China). Available soil P (AP) was extracted with 0.03 M ammonium fluoride-hydrochloric acid and measured colorimetrically as described above. Total K was determined using NaOH fusion method, and the available K (AK) was
Principal coordinates analyses (PCoA), based on Bray-Curtis dissimilarity, were used to display differences in the composition of bacterial communities between OJ and LP rhizosphere soil treatments. Permutational multivariate analysis of variance (PERMANOVA) was conducted to test the significance of the Bray-Curtis dissimilarity. Kruskal-Wallis tests were performed using the R software (kruskal. test function) to assess the impact of shade stress on soil bacterial community structure in both species. A value of P < 0.05 was considered to be statistically significant.
To analyze the correlations between soil physicochemical parameters and bacterial community compositions, a Mantel test (9,999 permutations) with Spearman correlations of the R vegan package was used. Canonical correspondence analyses (CCA) were performed with the R package vegan (v2.4.2) to visualize the relationship between soil physicochemical properties and bacterial communities. For the CCA analyses, the correlation of the canonical axes with the explanatory matrix was determined with the general permutation test and the "envfit" function was used to analyze the significance of soil physicochemical factors on the composition of bacterial communities. To analyze the correlations of above-and below ground phenotypes and the composition of bacterial communities, pairwise fitting analysis was carried out using the "lm" function in the R vegan package.
Additional file 2: Figure S1. Rarefaction curve of bacterial 16S rRNA gene sequences obtained from amplicon sequencing.
Additional file 3: Figure  Changes in the bacterial community composition at the phylum level (relative abundance > 1%, A), and genus level (relative abundance > 0.5%, B) under shade stress. Asterisks indicate statistically significant differences according to Kruskal-Wallis tests (*P < 0.05; **P < 0.01 and ***P < 0.001) Figure 5 Relationships between total leaf area (A), root volume (B), root surface area (C), Fv/Fm (D), and chlorophyll content (E) in bacterial community composition under shade stress. The relationship between total root length and bacterial community composition was not significant (data not shown). The plot shows the 95% confidence interval of the fit