To investigate the correlation between coloration and endophytic bacterial communities in blood orange pulp, this study employed microbiome analysis to compare the community structure and functional characteristics of endophytic bacteria between pigmented and non-pigmented blood orange pulp and it also evaluated their relationship with fruit quality. Results demonstrated that pigmented pulp exhibited significantly higher contents of total anthocyanins (38.78 versus 10.66 µg/g) and p-coumaric acid (0.59 versus 0.47 µg/g) and naringin (3.3 versus 1.53 µg/g) when compared with non-pigmented pulp. Microbiome profiling revealed richer bacterial diversity in pigmented pulp, containing 15 phyla, 25 classes, 46 orders, 89 families, 154 genera, and 87 species versus 11 phyla, 18 classes, 28 orders, 62 families, 103 genera, and 46 species in non-pigmented pulp. In pigmented pulp, Bacteroidetes was the dominant phylum, and Faecalibacterium, Bacteroides, Bifidobacterium, Prevotella, and Lactobacillus were the dominant genera. Correlation analysis showed that the differential Bacteroidetes species in pigmented pulp including Bacteroides vulgatus, B. caccae, and Parabacteroides goldsteinii had significantly positive correlations with anthocyanins, total flavonoids and naringin. PICRUSt2 functional prediction indicated endophytic bacterial gene functions were richer in pigmented pulp, with the significantly enriched functions mainly involved in post-synthetic modification (methylation), transport and storage of anthocyanins. These findings demonstrate that the quality of pigmented blood orange pulp is superior to that of non-pigmented blood orange pulp and that anthocyanin biosynthesis is closely related to endophytic bacteria such as B. vulgatus, B. caccae, and P. goldsteinii.

The composition and content of anthocyanins in a superior strain of ‘Tarocco’ blood orange and the ordinary variety were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS), and differential metabolites were selected. The results indicated that a total of 33 anthocyanin-related metabolites belonging to seven classes: cyanidin, delphinidin, peonidin, pelargonidin, malyidin, petunidin and flavonoid precursors of anthocyanin synthesis were identified in the superior strain. Furthermore, the number and kinds of anthocyanin metabolites in bore and peripheral fruits from the superior strain were identical, with no differential metabolites being found between them. In contrast, 27 and 30 metabolites were detected in bore and peripheral fruits from the control variety, and nine differential metabolites including cyanidin, delphinidin, peonidin and flavonoids were identified between bore and peripheral fruits. For both the superior strain and the ordinary variety, the main anthocyanin components were cyanidin-3-O-(6-O-malonyl)-glucoside and cyanidin-3-O-glucoside, accounting for 42.3% and 41.0% of the total anthocyanin content in the inner bore fruit and 40.6% and 41.4% of the total anthocyanin content in peripheral fruit from the superior line; and accounting for 39.5% and 41.0% of the total anthocyanin content in inner bore fruit and 36.8% and 40.6% of the total anthocyanin content in peripheral fruit from the common variety, respectively. Compared with the control variety, the amount of cyanidin and the proportion of total anthocyanins in the superior strain were significantly increased, and the total cyanidin contents in inner and peripheral fruits from the superior strain were 48.68 (91.90%) and 62.16 μg/g (92.28%), respectively. In addition, the percentage of delphinidin, peonidin and flavonoids in relation to the total anthocyanin content were similar between inner and peripheral fruits, suggesting that temperature may have little effect on anthocyanin synthesis in the superior strain. We concluded that the superior strain of ‘Tarocco’ blood orange has higher anthocyanin content than that the common variety and contains more kinds of metabolites. This study can provide a theoretical basis for genetic improvement and utilization of ‘Tarocco’ blood orange.