Sequencing data and microbial diversity
A total number of 1,555,170 fungal high-quality reads and 1,527,292 bacterial high-quality reads were generated by Illumina Novaseq 6000 sequencing, with an average of 129,598 fungal reads and 127,274 bacterial reads per sample (Table S1). After further quality control of the raw reads and removal of chimeras, the clean tags were clustered to obtain 2146 fungal OTUs and 4175 bacterial OTUs under a similarity threshold of 97% (Table S2). According to results from the experiment, the number of fungal OTUs was the highest at the BRB stage (34.4%), followed by the HR stage (23.5%) and BV stage (22.9%), while the number of bacterial OTUs was the highest at BV Stage (28.2%), followed by HR stage (25.7%) and BQR stage (23.3%). As can be seen from Fig. 1, there are 28 shared OTUs in the fungal communities and 118 shared OTUs in the bacterial communities during grape ripening. It shows that there are not only shared OTUs on the surface of the grape during the ripening process, but also specific OTUs at each ripening stage. At the same time, we found that the total amount of OTUs in the bacterial community was higher than that in the fungal community. Except that the number of fungal OTUs at the BRB Stage was higher than that of bacteria, the number of bacterial OTUs in other stages was higher than that of fungi, indicating that the species richness and diversity of the bacterial community were higher than that of the fungal community.
The Shannon and Simpson indices of the alpha-diversity index were used to reflect species diversity in fungal and bacterial communities, and the Chao1 and ACE indices were used to measure species richness (Table 1). Sequencing data showed that the coverage rate of fungi and bacteria community reached more than 99.8%, which makes clear that the quantity of the data was sufficient and could reflect the microbial community diversity of each mature stage. The Shannon diversity index showed that the fungal community had the highest diversity at the BV stage and the lowest at the HR stage, while the bacterial community had the highest diversity at the HR stage and the lowest at the BQR stage. The species richness of bacterial communities is highest at the BV and lowest at the HR stage. The diversity of the fungal community increased from the BRB stage to BV stage, and then decreased gradually, while the diversity of bacterial community decreased gradually from the BRB stage, but increased greatly approaching harvest ripening, there was no significant difference in the diversity of fungal and bacterial communities during maturation stages.
Table 1 Evaluation of species diversity and richness of fungal and bacterial communities at different stages of grape ripening
Ecolly Grape
|
Fungi
|
Bacteria
|
Maturation Stage
|
Sample ID
|
Shannon
|
Simpson
|
Chao1
|
ACE
|
coverage %
|
Shannon
|
Simpson
|
Chao1
|
ACE
|
coverage %
|
BRB
|
WEG1-1
|
3.3440
|
0.8138
|
266.05
|
271.64
|
99.96%
|
4.5316
|
0.9150
|
422.44
|
406.96
|
99.92%
|
WEG1-2
|
3.3756
|
0.8156
|
287.64
|
301.56
|
99.94%
|
4.3629
|
0.9030
|
436.97
|
432.81
|
99.92%
|
WEG1-3
|
3.5844
|
0.8308
|
496.76
|
490.95
|
99.90%
|
4.3801
|
0.9104
|
607.32
|
561.37
|
99.88%
|
BV
|
WEG2-1
|
4.5576
|
0.9406
|
200.05
|
204.94
|
99.94%
|
4.1172
|
0.8455
|
722.81
|
799.47
|
99.82%
|
WEG2-2
|
3.2503
|
0.7733
|
177.81
|
170.14
|
99.94%
|
4.0150
|
0.8370
|
614.74
|
626.51
|
99.86%
|
WEG2-3
|
3.4598
|
0.8496
|
228.00
|
228.83
|
99.93%
|
4.0896
|
0.8408
|
782.85
|
698.39
|
99.85%
|
BQR
|
WEG3-1
|
4.1221
|
0.9233
|
192.75
|
177.23
|
99.95%
|
2.6844
|
0.5929
|
650.76
|
694.73
|
99.85%
|
WEG3-2
|
2.9405
|
0.7771
|
167.67
|
139.57
|
99.95%
|
5.8616
|
0.9684
|
586.07
|
503.30
|
99.91%
|
WEG3-3
|
3.9211
|
0.9227
|
169.50
|
167.90
|
99.95%
|
2.7506
|
0.5981
|
520.40
|
529.55
|
99.87%
|
HR
|
WEG4-1
|
3.0063
|
0.8153
|
230.68
|
245.81
|
99.94%
|
4.6748
|
0.9313
|
456.10
|
447.17
|
99.90%
|
WEG4-2
|
3.7589
|
0.8668
|
178.27
|
176.25
|
99.96%
|
4.6615
|
0.9294
|
501.02
|
505.80
|
99.90%
|
WEG4-3
|
3.0912
|
0.8317
|
205.25
|
215.01
|
99.92%
|
4.7162
|
0.9310
|
488.00
|
494.83
|
99.91%
|
Microbial community structure
The microbial ecosystem on the surface of grape during ripening is evolving dynamically, and community substitution can have an important effect on microbial community habitat and grape quality. Principal Coordinate Analysis (PCoA) based on Unweighted Unifrac distance index was used to study sample relationships and microbial community structure differences during the mature stage of berries (Fig. 2). The results displayed that the contribution rates of PCo1 and PCo2 in fungal communities were 30.59% and 22.16%, respectively, with a total interpretation of 52.75%, and the contribution rate of PCo1 and PCo2 in bacterial communities was 20.03% and 17.31%༌with a total interpretation of 37.34%, respectively.
The fungal community samples in each mature stage were separated, and there were significant differences in the structure of the fungal community (Anism, R = 0.898, P = 0.001). The distribution of fungal samples at the BRB stage has a certain distance compared with other stages, and the community structure is quite different from other stages. On the contrary, the fungal samples from the HR and BV stages are relatively close and have similarities in the community structure. In addition, some fungal sample points indicate that the community structure in the BV, BQR, and HR stages are similar, reflecting the succession of the fungal community structure.
The results also showed that the differences in bacterial community structure during the maturation stages are smaller than those of fungi, but the bacterial communities in each stage also reflect moderate differences (Anism, R = 0.676, P = 0.001). It should be noted, however, that the bacterial community structure evolved during the pre-harvest maturation stage in the direction of PCo1, but the samples from the HR stage were relatively close together with that of BRB, and the community structure may have changed significantly during the late maturation.
Microbial community composition during maturation stages
Fungal community composition
Except for unclassified groups, all fungi samples belonged to 4 phyla, 132 families, and 210 genera. At the phyla level, Ascomycota, Basidiomycota, Mucoromycota, and Mortierellomycota were found, and Ascomycota and Basidiomycota were the main fungal phyla in the maturation stages (Fig. 3a). The relative abundance of Ascomycota was dominant in the pre-harvest ripening stages but decreased from 99.8% at the BRB stage to 70.5% at the BQR stage and 46.7% at the HR stage. Inversely, the relative abundance of Basidiomycetes showed a gradually increasing trend during the maturation process. Although the abundance was less than 1% during the BRB stage, it continued to increase thereafter, and the relative abundance reached 52.1% during the HR stage, becoming the dominant fungal genus.
At the fungal genera level, the top 8 genera in relative abundance include Alternaria, Naganishia, Filobasidium, Aureobasidium, Fusarium, Acremonium, Aspergillus, and Styachybotrys (Fig. 3c). It can be seen that Alternaria is the dominant fungal genus on the grape surface before harvest ripening, but its relative abundance continues to decrease throughout the maturation, from 36.8% at the BRB stage to 15.4% at the HR stage. We also observed that the relative abundance of Naganishia, Filobasidium, and Aureobasidium increased gradually during maturation, reaching 26.3%, 24.8%, and 7.1% respectively at the HR, which distinctly became the dominant fungal microbiota. In addition, Aspergillus mainly existed at the BV stage with a relative abundance of 5.93%, while a small amount of Fusarium was detected at the BQR stage with a relative abundance of 8.9%.
Bacterial community composition
All bacterial samples belong to 20 phyla, 210 families, and 404 genera except for unclassified taxa. The bacterial phyla with the highest relative abundance rankings were Proteobacteria, Firmicutes, Bacteroidetes, and Actinobacteria (Fig. 3b). Proteobacteria was the dominant bacteria in each stage, the relative abundance reached 65.9%-93.8%, and the number of Firmicutes was relatively high during the BQR stage, and the relative abundance reached 23.8%. Bacteroides and Actinomycetes existed in a small amount before grape ripening, but Bacteroidetes disappeared at the HR stage.
At the bacterial genus level, the top 8 genera in relative abundance are Sphingomonas, Brevundimonas, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, Massilia, Roseomonas, Methylobacterium, Caulobacter, and Luteimonas (Fig. 3d). The distribution of the bacterial community on the grape surface during the BRB stage was relatively uniform, and Brevundimonas (22.2%), Sphingomonas (13.5%), Roseomonas (10.8%), and Luteimonas (8.9%) were detected. However, the relative abundance of Sphingomonas during the BV and BQR periods reached 46.3% and 42.8%, which had a clear advantage before harvest. In addition, there were also some Methylobacteria (6.4%) and Massilia (6.7%) in these two stages. At harvest ripening, Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium, which had previously been less abundant, suddenly became the dominant genus, with a relative abundance of 26%, and some bacterial genera such as Brevundimonas (14.3%), Sphingomonas (10.4%), and Caulobacter (8.7%) were still existing.
Correlation analysis of core fungal and bacterial genera
The shared genera in the microbial community that rank the top 10 in relative abundance are defined as the core microbiota on the berry surface, with 21 shared genera of fungi and 63 shared genera of bacteria present during ripening stages were found (Fig. 4a, b) (Table S3). The relative abundance of related core genera shows significant differences, and Alternaria and Sphingomonas are the most important core fungi and bacteria during mature stages and they are not only ubiquitous, but also abundant (Fig. 4c, d). Other core fungal genera include Naganishia, Filobasidium, Aureobasidium, Fusarium, Aspergillus, Comoclathris, Candida, Chaetomium, and Penicillium, while other core bacterial genera include Brevundimonas, Allorhizium-Neorhizobium-Pararhizobium-Rhizobium, Massilia, Roseomonas, Methylobacterium, Caulobacter, Ralstonia, Chryseobacterium, and Acinetobacter.
The association between core fungi and bacteria was analyzed using an O2PLS model to investigate the interactions between core microbiota that play a major role in grape maturation (Fig. 5). Biplot is a centralized display of the score scatter plot and loading scatter plot, which can be used to judge whether the combination of the two sets of data can reflect the characteristics of samples and to judge the relevance and influence degree of different core genera. Biplot combined with the correlation matrix (Table S4) shows that the distribution of fungal genera is scattered and mainly negatively correlated, but the negative correlation is weak, whereas the three fungal genera, Filobasidium, Naganishia, and Aureobasidium, all clustered together, have a stronger positive correlation, while the negative correlation between Aureobasidium and Comoclathris was the strongest. For Bacteria genera, the distance between Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium and Caulobacter is the closest, and the positive correlation between them is more than 0.99. Ralstonia and Acinetobacter had a positive correlation of 0.98, and the same was true for Roseomonas and Chryseobacterium. However, the two bacteria with the highest relative abundance, Sphingomonas, and Brevundimonas, showed the strongest negative correlation relationship in bacterial community, but the degree of negative correlation was only 0.63.
Figure 5 also reflects the correlation between the fungal genera and the bacterial genera. We can observe that Fusarium and Massilia are relatively close in position, and the positive correlation between them reaches 0.86. Moreover, the correlation between the fungal genus Filobasidium and two bacterial genera including Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium and Caubacloter reached 0.9, showing a high positive correlation. However, the negative correlation between Filobasidium and Methylobacterium is greater than 0.6, which may show a certain antagonistic effect. On the whole, the positive correlation between the microbial community is stronger than the negative correlation, and the synergy among some genera may be more significant.
Functional prediction of microbial communities
The fungal and bacterial OTUs were compared to the FUNGuild and PICRUSt2 databases to evaluate the potential function information of fungal and bacterial communities at different maturity stages. The results indicated that FUNGuild provides 7 trophic and 51 guild analyses of the fungal community at different stages, and Animal pathogens-endophytes-phytopathogens-wood saprophytic fungi are common fungal community functions during the maturation stage, but their relative abundance decreases gradually (Fig. 6). Conversely, the functional abundance of Undefined Saprotroph and Animal Pathogen-Endophyte-Epiphyte-Plant Pathogen-Undefined Saprotroph showed an increasing trend, and the functional abundance was higher at the HR stage. At the same time, it is found that the functional abundance of Animal Pathogen-Endophyte-Fungal Parasite-Plant Pathogen-Wood Saprotroph and Wood Saproprotroph was higher at the BRB stage, and Animal Pathogen was the main functional type of the fungal community during the BV stage. However, Undefined Saprotroph is the main functional one of the fungal communities in the HR stage, and the changes of the fungal community function in different maturation stages affect grape berries quality and health.
Based on 16S rRNA, the PICRUSt2 database was used to predict the function of bacterial communities, and 32 functional types of Level 2 in bacterial communities were found at different ripening stages (Fig. 7). The relative abundance of the main bacterial functional types showed a trend of first decreasing and then increasing as grape mature. Metabolism is the main functional type of bacterial communities, mainly including amino acid metabolism, carbohydrate metabolism, and metabolism of cofactors and vitamins, and there is also the metabolism of terpenoids and polyketides, lipid metabolism and energy metabolism, etc. In addition, some bacterial functions are important at different times, such as Replication and Repair, Cell Motility, and Signal Transduction are more abundant at the HR stage, while the relative abundance of Transport and catabolism is lowest at HR and highest at the BRB stage. At the same time, it was found that Environmental adaptation had the lowest functional abundance during the BV stage and the highest at the HR stage.