Apple scion cultivar influence plant growth and soil property
Among apple scion-rootstock combinations, the plant height ranged from 3.22 m to 5.26 m, trunk circumference ranged from 23.48 cm to 42.85 cm, and leaf SPAD value ranged from 55.72 to 60.78, but did not vary among rootstock genotypes when grafted ‘Fuji’ and ‘Golden Delicious’, respectively (Table S2). On average, apple scion cultivar was the main effect factors of plant growth: plant height (P = 0.002), trunk circumference (P < 0.001), and SPAD value (P < 0.001) were differentially produced by scion cultivars (Table S3). In leaves, all non-structural carbohydrates, except sorbitol, varied between apple scion cultivars: starch (P < 0.001), fructose (P < 0.001), glucose (P < 0.001), and sucrose (P < 0.001) (Table S5). Starch, fructose and glucose concentrations were lower for ‘Fuji’ compared to ‘Golden Delicious’, but ‘Fuji’ had higher sorbitol and sucrose concentrations versus ‘Golden Delicious’ (Fig.1a). Starch (P < 0.001) and fructose (P < 0.001) differed among rootstock genotypes, especially ‘Fuji’ as scion (Table S4, 5). Scion/rootstock interactions were significant for starch and sucrose (Table S5). In roots, fructose, sorbitol, and sucrose were differed among rootstock genotypes and between scion cultivars and had significant scion/rootstock interactions (Table S4, S5). All the various parameters expressed greater concentrations in the ‘Fuji’ versus ‘Golden Delicious’ (Fig.1b).
A range of soil characteristics were determined for four replicates rhizosphere soil per scion-rootstock combinations and bulk soil samples. As expected, lower levels of pH were detected in rhizosphere soil than that in bulk soil (deduced by 3.12-4.93%), whereas rhizosphere soil had higher levels of soil organic matter, AN, AP and AK compared to bulk soil, increased by 81-131.9%, 29.92-68.20%, 19.30-66.83% and 73.01-148.05%, respectively (Fig. S2). Furthermore, all soil traits, except AN, were significantly influenced by scion cultivars (Table S6), of which, rhizosphere soil pH of ‘Fuji’ was significantly lower compared to ‘Golden Delicious’ (Fig.2a), contrary, ‘Fuji’ rhizosphere soil nutrition was higher as compared to ‘Golden Delicious’, especially AN (Fig.2). Additionally, all soil properties, except AK, were varied among rootstock genotypes and their interactions (Table S6).
Diversity of rhizosphere bacterial community are modified by the apple scion cultivar
To investigate the microbial composition and diversity of apple grafted combinations rhizosphere, the V3-V4 regions of the bacterial 16S rRNA gene were sequenced on the Illumina MiSeq PE300 platform. In total, we obtained 3 758 432 high-quality sequence from 56 samples (average, 67 114, range, 26 334 to 149 423 per sample) and 9 311 operational taxonomic units (OTUs) were identified (Table S7).
We found that the rhizosphere bacterial evenness (designed as Shannon index and phylogenetic diversity) and richness (represented by Chao1) did not significant differ among rootstock genotypes when grafted with ‘Fuji’ or ‘Golden Delicious’, But rhizosphere bacterial diversity differed between scion cultivars (Shannon: P < 0.01, phylogenetic diversity: P < 0.001, and Chao1: P < 0.01, Fig. 3). In addition, there was a significant scion/rootstock interaction for Shannon index (P = 0.038). All these diversity indices were significantly higher in the ‘Golden Delicious’ rhizosphere soils than those in ‘Fuji’ (Fig. 3).
Principal coordinate analysis (PCoA) of pairwise Bray-Curtis similarity matrices were performed to investigate rhizosphere bacterial beta diversity. The results revealed that rhizosphere bacteria of scion-rootstock combinations formed two distinct clusters, which separated along the second coordinate axis (Fig. 4a). Additionally, we found that scion cultivar explained 21.1% of total variance (p < 0.001, PERMANOVA), and a significantly interaction between scion cultivar and rootstock genotype, explained 16.6% of total variance (P = 0.002, PERMANOVA), in addition to the rootstock genotype (12.0%, P = 0.321, PERMANOVA, Table 1), suggesting that the largest source of variation in the rhizosphere bacteria was the different scion cultivars. Subsequently, we divided all samples into two groups according to scion cultivar and analyzed separately. The results showed that the apple rootstock genotype explained 37.6% in combinations grafted with ‘Fuji’ (P = 0.013, PERMANOVA, Fig. 4b) and 32.7% in combinations grafted with ‘Golden Delicious’ (P = 0.093, PERMANOVA, Fig. 4c). Pair-wise post hoc test comparison using PERMANOVA on the Bray Curtis similarity matrices, we found that the rhizobacterial community were similar among the rootstocks (grafted with ‘Fuji’), except M. xiaojinensis and M.hupehensis, and rhizobacterial structure did not differ among rootstock genotypes when grafted with ‘Golden Delicious’ (Fig. S3). We also used weighted UniFrac distances to measure the effect of scion cultivar on the bacterial community. The results observed with Bray-Curtis metrics, rhizosphere bacterial diversity revealed a significant difference between ‘Fuji’ and ‘Golden Delicious’ (Fig. S4).
Rhizosphere bacterial assembly is regulated by grafting different scion cultivars
Next, we examined variation in the rhizosphere bacteria of grafted combinations. According to the classification of OTUs, the apple scion-rootstock combinations rhizosphere harbored 49 bacterial phyla. The most dominated bacterial phyla were Proteobacteria, Acidobacteria, Actinobacteria, Chloroflexi, and Firmicutes (relative abundance added up to 79-88%) in all samples (Fig. S5a). Manhattan plots showed that 1,309 OTUs were dramatically different between two scion cultivars, mostly belonging to a wide range of bacterial phyla, including Proteobacteria, Acidobacteria, and Actinobacteria (Fig. 5a). The relative abundance of Proteobacteria, Acidobacteria, Gemmatimonadetes, and Planctomycetes significantly differed between scion cultivars, but did not show strikingly difference among rootstock genotypes (Fig. 5b, Fig. S5a). Conversely, Saccharibacteria has differently abundance among rootstock genotypes grafting the same scion cultivar (Fig. S5a, Table S8). While Actinobacteria and Verrucomicrobia relative abundance differed between scion cultivars and among rootstock genotypes, and there was a significant interaction between scion cultivar and rootstock genotype (Table S8). To further dissect the difference bacterial composition between scion cultivars, we found that among of the dominant bacterial phyla, Proteobacteria, Actinobacteria, and Firmicutes showed greater relative abundance in ‘Fuji’ than that in ‘Golden Delicious’ (Fig. 5b). On the contrary, the abundance of Acidobacteria were found specifically enhanced in rhizosphere of rootstocks grafted ‘Golden Delicious’ (Fig. 5b).
To acquire the best discriminant performance of taxa across grafted combinations, we classified the relative abundances of bacterial taxa in the genus level, the most abundant bacteria (relative abundance > 1.0%) were Pseudomonas, Bacillus, Steroidobacter, Pseudarthrobacter, Streptomyces, RB41, Sphingopyxis, Rhizobium, Acidibacter, and Bradyrhizobium (Fig.S5b). Out of ten dominant bacterial genera, all differed between two scion cultivars, except Pseudomonas (Fig. 5c). Out of the dominant bacteria genera, Bacillus, RB41, and Rhizobium only varied between two scion cultivars, Steroidobacter, Streptomyces, Sphingopyxis, and Bradyrhizobium differed between scion cultivars and among rootstock genotypes, and there was a significant interaction between scion cultivar and rootstock genotype (Table S9). Additionally, all except RB41 in the rootstock rhizosphere were remarkably increased from grafted ‘Fuji’ to grafted ‘Golden Delicious’, especially Bacillus, Streptomyces, Sphingopyxis, and Rhizobium (Fig. 5c). For instance, ‘Fuji’ had a high relative abundance of Bacillus, Streptomyces, Sphingopyxis, and Rhizobium (RA 4.2%, 2.85%, 1.90%, and 1.53%, respectively), which were approximately twice as much as that in ‘Golden Delicious’ (RA 1.98%, 1.21%, 0.84%, and 0.89%, respectively) (Fig. 5c). The above results indicated that apple scion cultivars as above the ground parts of scion-rootstock combinations had a significant regulatory impact on the rhizosphere bacterial diversity and taxonomic composition (Table 1).
Table 1 PERMANOVA on the Bray-Curtis similarity matrices estimated the components of factor affect rhizosphere bacterial beta diversity in apple scion-rootstock combinations.
Factor
|
Sum Sq
|
R2
|
F
|
Pr (>F)
|
Scion cultivar
|
0.50841
|
0.2106
|
17.5876
|
< 0.001
|
Rootstock genotype
|
0.2907
|
0.12
|
1.1181
|
0.321
|
Interaction
|
0.40085
|
0.166
|
2.3111
|
0.002
|
Residual
|
1.21411
|
0.503
|
|
|
Total
|
2.41407
|
1
|
|
|
Spearman correlations between the root sugar and rhizosphere bacterial communities
Spearman's rank correlation analysis was conducted to assess the effect of root sugars in shaping the rhizosphere bacterial assembly (Fig. 6). As showed in the heatmap, fructose in root was positively correlated with Actinobacteria and negatively correlated with Acidobacteria, Elusimicrobia, and Gemmatimonadetes. Root sucrose showed the greatest positive correlation with the abundance of Actinobacteria, Saccharibacteria, and Cyanbacteria, and was negatively correlated with the abundance of Acidobacteria, Gemmatimonadetes, Nitrospirae, Elusimicrobia, and Latescibacteria. Sorbitol showed the positive correlation with the abundance of Actinobacteria. Moreover, Saccharibacteria and Nitrospirae presented, respectively, the positive and negative correlation with starch in root (Fig. 6a). At bacterial genus level, all except Preudarthrobacter showed the positive correlation with sugars in root, being especially positively correlated with sucrose (Fig. 6b). The abundance of Streptomyces, Sphingopyxis, and Sphingomonas were positively correlated with root fructose and sorbitol. Moreover, Rhizobium and Acidibacter were significantly positively correlated with Fructose and starch, respectively (Fig. 6b).
Apple scion cultivars drive the rhizosphere bacterial functional metabolism
Using PICRUSt as a predictive exploratory tool to establish if the expression of rhizosphere bacterial functions were correlated with the apple scion cultivars. We found that 41 level 2 KEGG Orthology groups (KOs) were represented in the rhizosphere data set (Fig. S6), and 22 KEGG pathways were significantly different abundance between ‘Fuji’ and ‘Golden Delicious’ (Fig. 7a). Nine KEGG pathways were significantly greater in rhizosphere of ‘Fuji’; by contrast, ‘Golden Delicious’ rhizosphere exhibited enrichment for 13 KEGG pathways (Fig. 7a). Importantly, the pathways of carbohydrate metabolism and energy metabolism represented about 16% of all the results, and ‘Fuji’ exhibited a greater abundance of carbohydrate metabolism and lower abundance of Energy metabolism than those of ‘Golden Delicious’ (Fig. 7a). A sophisticated analysis of the functional groups revealed that the most enriched functional subfunctions of Carbohydrate metabolism were influenced by apple scion cultivars, which including fructose and mannose metabolism, starch and sucrose metabolism, and galactose metabolism (Fig. 7b). Additionally, bacterial community plant growth-promoting (PGP) traits (such as nitrogen metabolism, sulfur metabolism, and inositol phosphate metabolism) were spread throughout the bacterial communities associated with the apple scion cultivars (Fig. 7b, c).