Six biological replicates each were sampled from four fruit species (blueberries, cherries, raspberries, and strawberries) at four developmental stages. Developmental stages were based on fruit pigmentation ranging from unripe (green) to fully ripe (red/purple/navy; Fig. S1) throughout June to September in 2018. Ten fruits (except blueberries N = 20) were collected for each species per replicate, and this was replicated six times for each ripening stage for each fruit at different sites.
Quantitative analysis of fungal communities
Metabarcoding analysis is generally not quantitative, but the addition of 265 P. cucumerina cells to sub-samples prior to DNA extraction served as an internal standard to attempt an estimation of the size of fungal populations. One replicate spiked with the internal standard of the strawberry stage 3 samples was removed due to poor sequence quality leaving 96 non-spiked and 95 spiked samples which produced a total of 38,445,395 reads that clustered into 1712 > 97% identity Amplicon Sequence Variants (ASV), which from here-in we call phylotypes (Table S1). Blast searches across all phylotypes for matches to the P. cucumerina internal standard’s ITS sequence generated from Sanger sequencing revealed one phylotype that matched with 100% identity. Plectosphaerella cucumerina was naturally present in 21 of the 95 non-spiked samples and comprised of a total of 444 reads. Cherry was the only fruit where the internal standard was reliably recovered: 23 of 24 spiked samples and only one of 24 non-spiked samples contained the internal standard phylotype. After internal standard DNA read normalisation, the mean (± SE) number of fungal cells from each of the useable 23 pairs of cherry replicates was 307,323 (± 39,090) cells. The range of phylotype cell abundance across all cherry samples was 3.9 million for an Aureobasidium phylotype to 3 cells for a phylotype taxonomically assigned no higher level than kingdom. There was no significant change in total fungal cell numbers across cherry maturation stage (Kruskal–Wallis, chi-squared = 2.63, P = 0.45; Fig. S2), but fruit surface areas also increased significantly (Kruskal–Wallis, chi-squared = 19.70, P = 0.0002, Fig. S2). When cell numbers were normalised for surface area this revealed that absolute fungal population sizes remained static across cherry maturation stages (Kruskal–Wallis, chi-squared = 2.49, P = 0.48; Fig. 1A). However, there was a significant change in absolute Saccharomycetales cell numbers when normalised for cherry surface area across maturation (Kruskal–Wallis, chi-squared = 15.30, P = 0.002): stage 1 had significantly greater absolute Saccharomycetales cell numbers than stage 4 (P = 0.0007; Fig. 1B). Six individual Saccharomycetales yeast phylotypes from the genera Debaryomyces, Saccharomyces, Kodamaea, one from the family Pichiaceae, and phylotypes with > 97% homology to M. pulcherrima and Metschnikowia gruessii, had significantly greater abundances on ripening stage 1 than 4 (P values span 0.045 to 0.006).
Absolute fungal cell abundances on cherry epicarp. Number of total fungal (A) and Saccharomycetales yeasts (B) cells per mm2 of cherry epicarp (N = 6 except, stage 3 and 4, N = 5) at four ripening stages (1, unripe/green fruit; 2, de-greening fruit; 3, ripening fruit; and 4, fully ripe/harvest fruit) estimated from DNA read abundances normalised to DNA abundances from the deliberate addition of 265 live Plectosphaerella cucumerina cells prior to DNA extraction. Different lower-case letters above bars show significant differences between ripening stages at P > 0.05, Dunn’s comparisons post-hoc with Benjamini–Hochberg multiple comparison correction.
Overview of fungal diversity across all fruit samples
The P. cucumerina internal standard phylotype was removed from all samples, and the sequence data normalised and analysed. A total of 1712 fungal phylotypes was revealed, comprising seven phyla, 25 classes, 96 orders, 197 families, and 280 genera. The most abundant and diverse phylum was Ascomycota, comprising 92.2% of reads and 57.3% of phylotypes, followed by Basidiomycota (7.7% reads and 33.6% phylotypes), Zygomycota (0.1% and 1.1%), Chytridiomycota (> 0.1% and 0.7%), Mucoromycota (> 0.1% and 0.3%), Glomeromycota and Rozellomycota (both > 0.1% and 0.1%; Fig. S3A). A phylotype from the Cladosporium genus was the most common phylotype across all samples, comprising 60.8% of reads. A total of 87 phylotypes from the order Saccharomycetales (budding yeasts) was detected, comprising 1,792,782 DNA reads (4.7% of the total) spanning 10 families and 25 genera. Metschnikowia was the most abundant Saccharomycetales genus (40.0% of Saccharomycetales reads), followed by Hanseniaspora (38.2%), then Pichia (5.2%), with the remaining genera contributing fewer than 3% each. Candida was the most diverse genus within the order Saccharomycetales accounting for 21.8% of phylotypes, despite only comprising 2.4% of reads, followed by Metschnikowia (11.5%), Hanseniaspora (8.0%) and Pichia (6.9%), with each of the remaining genera contributing fewer than 3.5% of phylotypes each (Fig. S3B). The most common Saccharomycetales yeast across all samples was a phylotype from the genus Hanseniaspora with > 97% homology to H. uvarum and comprised 38.2% of the total Saccharomycetales reads (Fig. S3B).
The effect of fruit species and ripening stage on epicarp fungal communities
We analysed differences in three biodiversity metrics to evaluate the effect of fruit species and maturation stage on fungal communities: differences in the absolute numbers of phylotypes (richness); differences in the types of phylotypes (i.e. presences/absences); and differences in the relative abundances of phylotypes (community composition) following Morrison-Whittle et al.14 and Morrison‐Whittle and Goddard37.
Fungal phylotype richness
Phylotype richness was not normally distributed (Shapiro-Wilks, P = 0.008) but square root transformation allowed the data to conform to the assumptions of ANOVA. There was a significant effect of both fruit type and ripening stage on the number of fungal phylotypes, including an interaction between the two (F3,175 = 18.58, P = 1.65 × 10–10; F3,175 = 5.00, P = 0.002 and F9,175 = 6.69, P = 3.25 × 10–8 respectively). Comparisons of effect sizes revealed fruit type (ω2 = 0.30) had a 4.4 times greater effect than ripening stage (ω2 = 0.068) on fungal phylotype richness. Disregarding ripening stage, cherry (mean ± SE number of phylotypes = 98 ± 4.1) had significantly more fungal phylotypes than blueberry (68 ± 3.7), raspberry (72 ± 2.9) and strawberry (76 ± 3.2) (Tukey’s HSD, P < 1.0 × 10–7, P = 2.0 × 10–7 and P = 2.56 × 10–5 respectively), which did not differ from one another (Fig. S4). Disregarding fruit type, ripening stage 2 (mean ± SE number of phylotypes = 85 ± 2.9) and 3 (82 ± 4.1) had significantly more fungal phylotypes than stage 1 (P = 0.001 and P = 0.033, respectively), but numbers at stages 1 and 4 were not significantly different (Fig. S4). The absolute time points for sampling did however differ between fruits due to different maturation timings.
As there was a significant interaction between fruit and ripeness stage, we investigated the effect of ripening stage on each fruit separately. All data residuals were normally distributed (Shapiro–Wilks, P > 0.05) and there was a significant effect of ripening stage on the number of fungal phylotypes for cherry, raspberry, and strawberry (one-way ANOVA: F3,44 = 4.33, P = 0.0093; F3,44 = 13.56, P = 2.11 × 10–6 and F3,44 = 13.86, P = 1.84 × 10–6, respectively, Fig. 2), but not blueberry (F3,44 = 2.27, P = 0.055). On cherries phylotype numbers increased during ripening, but raspberry and strawberry had greater numbers at intermediate stages of fruit maturation (Fig. 2).
Number of observed phylotypes across fruit types and maturation stages. Number of fungal phylotypes across four ripening stages (1, unripe/green fruit; 2, de-greening fruit; 3, ripening fruit; and 4, fully ripe/harvest fruit) for blueberry, cherry, raspberry and strawberry (N = 12 except N = 11 for strawberry stage 3). Numbers of fungal phylotypes differ across ripening stages for cherry, raspberry and strawberry but not blueberry (ANOVA, P values shown). Where significant, different lowercase letters represent significant differences in phylotype numbers within each fruit (P < 0.028) with separate Dunn’s comparisons post-hoc (with Benjamini–Hochberg multiple comparison correction). Different letter groups show any significant differences between ripening stages within each fruit separately.
There was a significant effect of fruit type but not ripening stage on the number of Saccharomycetales budding yeast phylotypes (Kruskal–Wallis, chi-squared = 75.66, df = 3, P = 2.61 × 10–16 and chi-squared = 5.50, df = 3, P = 0.14 respectively). Raspberry (mean ± SE number of phylotypes = 12 ± 0.60) harboured significantly more Saccharomycetales phylotypes than strawberry (10 ± 0.74), cherry (7 ± 0.70), and blueberry (4 ± 0.31; Tukey’s HSD, P = 0.044, P = 2.9 × 10–6 and P = 1.5 × 10–15 respectively). Strawberry harboured significantly more phylotypes than cherry and blueberry (Tukey’s HSD, P = 0.007 and P = 2.6 × 10–9), and cherry harboured significantly more than blueberry (Tukey’s HSD, P = 0.001) (Fig. S5). Both Shannon’s and Simpson’s diversity indexes, which analyse the distribution of phylotype abundances, revealed differences between fruit species and ripening stage in line with the above findings (Table S2).
Presence/absence of fungal phylotypes
Both fruit type and ripening stage significantly influenced the types of fungi present (PermANOVA, R2 = 0.094, P = 9.999 × 10–5 and R2 = 0.017, P = 9.999 × 10–5, respectively, Fig. 3A) and there was a significant interaction between fruit type and ripening stage (R2 = 0.013, P = 9.999 × 10–5). Comparisons of effect sizes (R2 values) showed fruit type had approximately 5.5 greater influence than ripening stage on the types of fungal phylotypes present. As there was a significant interaction between fruit and ripening stage, the effect of ripening stage on fungal communities was investigated for each fruit separately. Ripening stage significantly influenced the types of fungal phylotypes present on all fruit (blueberry R2 = 0.043, 9.999 × 10–5; cherry R2 = 0.060, P = 9.999 × 10–5; raspberry R2 = 0.13, P = 9.999 × 10–5 and strawberry R2 = 0.055, P = 9.999 × 10–5, Fig. S6). There were significant differences in presences of fungal phylotypes between all fruits and ripening stages (post-hoc pairwise PermANOVAs: P = 9.999 × 10–5, R2 range 0.09–0.20; Fig. 3A; Supplemental Tables S3, S4).
NMDS plots representing the differential presences of fungal phylotypes. Nonmetric Multidimensional Scaling (NMDS) plots of binary Jaccard measures of community dissimilarity of (A) total fungal communities and (B) Saccharomycetales budding yeasts on blueberry (blue), cherry (purple), raspberry (green) and strawberry (red) at four ripening stages (1, unripe/green fruit; 2, de-greening fruit; 3, ripening fruit; and 4, fully ripe/harvest fruit; denoted by shade of colour, lightest shade for green fruit and moving through to darkest shade for fully ripe/harvest). Both total fungal and Saccharomycetales yeasts communities significantly differ in the presences of phylotypes across all fruit types (FT) and ripening stages (RS) by PermANOVA (values shown top right).
Both fruit type and ripening stage significantly influenced the types of Saccharomycetales phylotypes present (PermANOVA, R2 = 0.082, P = 9.999 × 10–5 and R2 = 0.026, P = 9.999 × 10–5, respectively, Fig. 3B) with a significant interaction between fruit type and ripening stage (R2 = 0.024, P = 9.999 × 10–5). In line with the general fungal community, comparisons of R2 values showed fruit type had approximately 3.15 times greater effect than ripening stage on the Saccharomycetales phylotypes present. Ripening stage significantly influenced the types of Saccharomycetales phylotypes on each fruit separately (blueberry R2 = 0.065, P = 0.0008; cherry R2 = 0.080, P = 0.0004; raspberry R2 = 0.27, P = 9.999 × 10–5 and strawberry R2 = 0.084, P = 9.999 × 10–5). There were significant differences in presences of different Saccharomycetales yeast phylotypes between all fruits and ripening stages (post-hoc pairwise PermANOVAs: P = 9.999 × 10–5, R2 range 0.06–0.15; Supplemental Tables S5, S6).
Relative abundances of fungal phylotypes
Fruit type and ripening stage also significantly influenced the relative abundances of different fungal phylotypes (PermANOVA, R2 = 0.15, P = 9.999 × 10–5 and R2 = 0.027, P = 0.0002, respectively, Fig. 4A), and the interaction between them was also significant (R2 = 0.018, P = 0.003). Fruit type had approximately 5.6 times greater influence than ripening stage on the relative abundances of fungal phylotypes. Ripening stage significantly influenced the relative abundances of fungal phylotypes present on each fruit separately (blueberry R2 = 0.16, P = 9.999 × 10–5; cherry R2 = 0.061, P = 0.009; raspberry R2 = 0.24, P = 9.999 × 10–5 and strawberry R2 = 0.15, P = 9.999 × 10–5, Fig. S7). There were significant differences in fungal community composition between all fruits and ripening stages (post-hoc pairwise PermANOVAs: P = 9.999 × 10–5, R2 range 0.11–0.57; Supplemental Tables S7, S8).
NMDS plots representing the differential abundances of fungal phylotypes. Nonmetric Multidimensional Scaling (NMDS) plots of abundance Jaccard measures of community dissimilarity of (A) total fungal communities and (B) Saccharomycetales budding yeasts on blueberry (blue), cherry (purple), raspberry (green) and strawberry (red) at four ripening stages (1, unripe/green fruit; 2, de-greening fruit; 3, ripening fruit; and 4, fully ripe/harvest fruit; denoted by shade of colour, lightest shade for green fruit and moving through to darkest shade for fully ripe/harvest). Both total fungal and Saccharomycetales yeasts communities significantly differ in the presences of phylotypes across all fruit types (FT) and ripening stages (RS) by PermANOVA (values shown top left).
Fruit type and ripening stage significantly influenced the relative abundances of Saccharomycetales phylotypes (PermANOVA, R2 = 0.038, P = 9.999 × 10–5 and R2 = 0.024, P = 9.999 × 10–5, respectively, Fig. 4B), with an interaction between the main effects (R2 = 0.016, P = 9.999 × 10–5). Fruit species had approximately 1.6 times greater influence than ripening stage on the relative abundances of phylotypes. Ripening stage significantly affected the relative abundances of Saccharomycetales phylotypes on each fruit separately (blueberry R2 = 0.043, P = 0.004; cherry R2 = 0.64, P = 0.003; raspberry R2 = 0.19, P = 9.999 × 10–5 and strawberry R2 = 0.070, P = 0.0009). There were significant differences in Saccharomycetales community composition between all fruit species and ripening stages (post-hoc pairwise PermANOVA: P = 9.999 × 10–5, R2 range 0.038–0.10; Supplemental Tables S9, S10).
The similarities and differences of fungal phylotypes
The core fruit fungal microbiome
Analyses thus far have focussed on differences in fruit microbiomes, but it is valuable to contrast this with quantifying fruit microbiome similarity. The core fruit fungal microbiome (i.e. those phylotypes present across all fruits) consisted of 199 (11.6%) of the 1712 fungal phylotypes and comprised 97.6% of DNA reads (Table S11). Approximately 12–22% of the 1712 phylotypes were only found associated with specific fruits: 216 with blueberry, 372 with cherry, 201 with raspberry, and 242 with strawberry (Fig. 5A, Table S11). Twenty of the 87 Saccharomycetales phylotypes (23.0%) comprising 81.2% of Saccharomycetales reads were present across all fruit types (Table S11), with 3 unique to blueberry, 5 to cherry, 25 to raspberry and 15 to strawberry (Fig. 5B, Table S12).
Comparison of shared and discrete numbers of fungal and Saccharomycetales phylotypes between fruits. Venn diagrams of phylotype counts across fruit types for (A) all fungal phylotypes, and (B) Saccharomycetales phylotypes; overlapping segments are approximately proportional to values.
The phylotypes that are most differentially abundant
Analyses across all biodiversity metrics show fruit type had a greater effect on fungal communities than maturation stage. Overall, 195 (11.4%) indicator phylotypes (spanning 76 families) had significantly differential abundances between fruit types: 33 phylotypes were significantly overrepresented on blueberry, 70 on cherry, 39 on raspberry and 53 on strawberry (FDR corrected P values ranging from P = 0.011 to P = 0.044). The complete list of significantly differentially overrepresented phylotypes is shown in Table S13 but the two most significantly differentially overrepresented phylotypes on each fruit are listed here: Polyphialoseptoria species and Ramularia (most likely Ramularia endophylla) on blueberry; Exobasidium species and a phylotype from the poorly described order Leotiomycetes on cherry; phylotypes with > 97% homology to Metschnikowia kunwiensis and H. uvarum on raspberry; and phylotypes with > 97% homology to Kalmanozyma fusiformata (Ustilaginaceae smut fungi) and Podosphaera aphanis on strawberry.
Twenty-four of the 195 indicator phylotypes belonged to the Saccharomycetales budding yeasts (Table S13). There were no Saccharomycetales indicator phylotypes for cherry, and just one for blueberry, a fungal phylotype with > 97% homology to Metschnikowia koreensis. Raspberry had 15 Saccharomycetales indicator phylotypes: three with > 97% homology to the Metschnikowia and, Candida genera, two Pichia and Schwanniomyces, and one each from Hanseniaspora, Barnettozyma, Debaryomyces, Candida, Geotrichum and Martiniozyma. There were eight indicator phylotypes for strawberry; two Candida and one from each of the Metschnikowia, Starmerella, Kodamaea and Hyphopichia genera and the Pichiaceae family, and a phylotype assigned to the no higher level than fungal kingdom (with > 97% homology to deposit from Candida genus). The dynamics of Saccharomycetales yeast indicator phylotypes abundances across maturation for raspberry and strawberry is shown in Fig. 6.
Dynamics of changes in the proportion of budding yeast indicator phylotypes. Mean proportion of reads for the Saccharomycetales budding yeast indicator phylotypes that are significantly overrepresented on (A) raspberry and (B) strawberry (P < 0.04) across the four ripening stages (1, unripe/green fruit; 2, de-greening fruit; 3, ripening fruit; and 4, fully ripe/harvest fruit). Indicator phylotypes are reported to the taxonomic level assigned: lower case letter refers to the taxonomic hierarchy of respective taxa (g = genus; f = family; k = kingdom). Where possible, assignment to genus taxonomic levels is shown in parentheses from matches to deposits in Genbank with > 97% homology identified by manual Blast searches.
Differences of yeast known to be attractive to D. suzukii
Yeast from the Hanseniaspora, Pichia, Saccharomyces, Candida and Metschnikowia genera and their combinations are attractive to D. suzukii27,28,30,31, and phylotypes belonging to these genera were recovered here. The combined relative read abundances of all phylotypes assigned to these genera were significantly different between fruit types and ripening stages (Kruskal–Wallis chi-squared = 60.54, P = 4.51 × 10–13; chi-squared = 10.11, P = 0.018, respectively). Raspberry had the highest relative abundance of yeast genera known to be attractive to D. suzukii (mean ± SE = 21,539 ± 4339) and this was significantly greater than on the other fruits (P < 1.97 × 10–8): cherry (1535 ± 265), strawberry (1651 ± 234) and blueberry (8009 ± 2648). When fruit types were analysed individually, ripening stage had a significant effect on relative read abundance of attractive yeast genera for raspberry only (Kruskal–Wallis chi-squared = 28.70, P = 2.59 × 10–6) where stage 1 and 4 abundances did not significantly differ (mean ± SE = 5682 ± 1522 and 20,826 ± 4711 respectively) but were significantly greater than stage 2 and 3 (2163 ± 538 and 4113 ± 1494 respectively; P < 0.05; Fig. S8).
Various isolates of H. uvarum have consistently been shown to be attractive to D. suzukii27,28,30,31,32. Seven phylotypes were assigned to Hanseniaspora and four of these had > 97% homology to H. uvarum deposits in Genbank using Blast searches, and the relative abundance of these four phylotypes significantly differed between fruit types (Kruskal–Wallis chi-squared = 70.67, df = 3, P = 3.08 × 10–15). Raspberry had the highest relative abundance of H. uvarum (mean ± SE 13,843 ± 3991) but this was not significantly greater (P = 0.080) than abundances on strawberry (426 ± 134) but was significantly greater than blueberry (6 ± 4) and cherry (8 ± 2; P = 1.62 × 10–12, P = 6.00 × 10–9 respectively). Raspberry and strawberry were the only fruits where maturation stage had a significant effect on H. uvarum relative abundance (Kruskal–Wallis chi-squared = 33.40, df = 3, P = 2.66 × 10–7; chi-squared = 12.59, df = 3, P = 0.006), and H. uvarum relative abundance increased as fruits ripened (Fig. S9). This analysis is in line with the indicator phylotype analysis which reported a Hanseniaspora phylotype with > 97% homology to H. uvarum as over-represented on raspberry generally, and especially at later stages (Fig. 6A).
Differences of Botrytis cinerea, known to be repulsive to D. suzukii
The relative read abundances of B. cinerea were significantly different between fruit types and ripening stages (Kruskal–Wallis chi-squared = 73.45, P = 7.80 × 10–16; Kruskal–Wallis chi-squared = 23.81, P = 2.74 × 10–5, respectively). Raspberry had the lowest relative abundance of B. cinerea (mean ± SE = 800 ± 136) and this was significantly lower than strawberry (1994 ± 292) and blueberry (5990 ± 1305) (P < 0.004), but not cherry (3015 ± 1406). Cherry and strawberry had significantly lower reads than blueberry (P < 4.13 × 10–5). When fruit types were analysed individually, ripening stage had a significant effect on relative read abundance of B. cinerea for all fruits (P < 0.003) and reads generally increased during ripening except on cherry where stage 2 had the greatest (Fig. S10).
Correlations with fruit host potential index (HPI) scores
Finally, Bellamy et al.38 generated fruit host potential index (HPI) scores from interactions of D. suzukii with commercial ripe fruit including the fruit species analysed here. The combined relative abundances (i.e. the total number of reads on each fruit across replicates) of yeast phylotypes empirically shown to be attractive to D. suzukii (Hanseniaspora, Pichia, Saccharomyces, Candida and Metschnikowia27,28,29,30,31) across these different ripe fruits at the last sample point are positively correlated with fruit HPI scores (Pearson’s correlation r = 0.38), as are the relative abundance of just H. uvarum (r = 0.62). The relative abundance of B. cinerea was negatively correlated to HPI scores (r = − 0.65) (Fig. S11); however, none of these correlations were significant (P > 0.35) likely due to the low number of comparisons (N = 4 due to just one HPI score per fruit type).
Source: Ecology - nature.com
