Variations in the Community Structure of Fungal Microbiota Associated with Apple Fruit Shaped by Fruit Bagging-Based Practice

Abstract

The various fungal communities that adhere to apple fruit are influenced by agricultural practices. However, the effects of fruit bagging-based management practice on the fungal microbiota are still unknown, and little is known about the fungal communities of bagged apple fruit. We conducted a study using apple fruit grown in a conventionally managed orchard where pesticide use is an indispensable practice. Fungal communities were collected from the calyx-end and peel tissues of bagged and unbagged fruit and characterized using barcode-type next-generation sequencing. Fruit bagging had a stronger effect on fungal richness, abundance, and diversity of the fungal microbiota in comparison to non-bagging. In addition, bagging also impacted the compositional variation of the fungal communities inhabiting each fruit part. We observed that fruit bagging had a tendency to maintain ecological equilibrium since Ascomycota and Basidiomycota were more distributed in bagged fruit than in unbagged fruit. These fungal communities consist of beneficial fungi rather than potentially harmful fungi. Approximately 50 dominant taxa were detected in bagged fruit, for example, beneficial genera such as Articulospora, Bullera, Cryptococcus, Dioszegia, Erythrobasidium, and Sporobolomyces, as well as pathogenic genera such as Aureobasidium and Taphrina. These results suggested that fruit bagging could significantly increase fungal richness and promote healthy fungal communities, especially the harmless fungal communities, which might be helpful for protecting fruit from the effects of pathogens. This study provides a foundation for understanding the impacts of bagging-based practice on the associated fungal microbiota.

Keywords: Malus domestica; bagged apple fruit; biodiversity; fungal community composition; metabarcoding; microbiome; richness.

Figure 1

The Venn diagrams show the total number of 3331 OTUs (97% sequence identity) shared across different samples, as well as the number of OTUs present exclusively in calyx-end and peel tissues derived from bagged (i.e., CR5, PFR5) and unbagged (i.e., CR3 and PFR3) fruit.

Figure 2

Bar charts (a) and heat map (b) showing the relative abundance of 18 fungal genera that were present across all samples. In the heat map, blue represents a fungus with relatively low abundance, and red represents a fungus with relatively high abundance. Cluster analyses of samples (Vertical) and genus classification units (horizontal) were performed according to the similarity of distributions.

Figure 3

Box-and-whisker plots, based on Chao1 (a), Shannon (b), and Simpson (c) diversity indices showing fungal richness and diversity in the calyx-end and peel tissues derived from bagged (i.e., CR5 and PFR5) and unbagged (i.e., CR3 and PFR3) fruit. The Chao1 index is commonly used to estimate the number of OTUs or species in samples. The Shannon and Simpson diversity indices are common measures of diversity, which reflect the richness and evenness of the samples.

Figure 4

Box-and-whisker plots visualizing the results of the analysis of similarity (ANOSIM) to estimate fungal communities associated with calyx-end and peel tissues of different management practices, i.e., bagging (CR5 and PFR5) and non-bagging (CR3 and PFR3). In the plot, the y-axis represents the distant rank between samples, and the x-axis represents the results among samples.

Figure 5

The correlation heatmap illustrates the relationship between each dataset of the calyx-end and peel tissues derived from bagged (i.e., CR5, PFR5) and unbagged (i.e., CR3 and PFR3) fruit and their replication compared to every other dataset. The red color indicates a close correlation, while the green color indicates a more distant correlation or given color key value exhibition ranging from 0 (complete distance) to 1 (complete close correlation) in the data.

Figure 6

Non-metric multidimensional scale (NMDS) plots corresponding to the clustering of fungal communities in different habitats, i.e., calyx-end tissue from unbagged fruit: CR3 (red); calyx-end tissue from bagged fruit: CR5 (green); peel tissue from unbagged fruit: PFR3 (blue); peel tissue from bagged fruit: PFR5 (purple). Cluster analysis was performed using the Bray–Curtis coefficient-based community similarity measure. In the plot, the x-axis and y-axis represent the 1st and 2nd principal coordinates, respectively. The percentage of the principal coordinates represents the relative contribution of the coordinate to the sample differences. A closer distance represents higher similarity, and samples that cluster together consist of similar microbiota.

Figure 7

LEfSe analysis illustrating the differences in the relative abundance of taxa of calyx-end and peel tissues of bagged apple fruit. (a) The linear discriminant analysis (LDA) histogram exhibits biomarkers of fungal microbiota of bagged apple fruit. The histogram shows 54 taxonomic clads of fungal taxa with significant differences (cut-off score ≥ 2.0). (b) The cladogram with significantly discriminant taxon nodes colored. Red and green circles mean that the taxa showed differences in relative abundance, and yellow circles mean non-significant differences; branch areas are shaded according to the highest ranked group for that taxon.

Figure 8

LEfSe analysis illustrating the differences in relative abundance of the taxa of calyx-end and peel tissues of unbagged apple fruit. (a) The linear discriminant analysis (LDA) histogram exhibits biomarkers of fungal microbiota of unbagged apple fruit. The histogram shows 10 taxonomic clades of fungal taxa with significant differences (cut-off score ≥ 2.0). (b) The cladogram with significantly discriminant taxon nodes colored. Red and green circles mean that taxa show differences in relative abundance, and yellow circles mean non-significant differences; branch areas are shaded according to the highest ranked group for that taxon.

Figure 9

Relative abundance (%RA) at the genus level of fungal traits of apple-inhabiting fungi associated with bagged fruit. Fungal property: apple pathogenic fungi (AP), beneficial fungi (BF), unknown (U).