This is the first study to provide a global analysis of the apple fruit (‘Royal Gala’) microbiome and determine the structure and diversity of microbial communities on and in different fruit tissues at harvest. A core microbiome shared between apple samples in all locations was identified suggesting that the members of the core microbiome may have co-evolved with the domestication of apple and potentially may play an essential role in defining fruit traits related to disease resistance and fruit quality. We characterized the microbial communities associated with ‘Royal Gala’ apple fruit at harvest maturity stage and assessed the effect of geographical location on both large-scale spatial variations, i.e. across different countries, and small-scale spatial variations, i.e. within a fruit. While the microbiome associated with plants has been extensively studied, knowledge about the fruit microbiome is still rather limited relative to rhizosphere, endophyte, and phyllosphere studies [14, 15]. Additionally, information about the role of the fruit microbiome on pre- and postharvest diseases, as well as fruit physiology, is also lacking. This is despite the importance of postharvest losses in reducing the economic return from fruit production, especially after so many resources have already been expended to produce a harvestable crop. Apples also encounter losses in storage, transit, markets, and homes due to postharvest pathogens . For over 30 years, there has been considerable research focus on the development of biological control strategies based on naturally-occurring microorganisms [16, 41]. Especially with the use of yeast antagonists, has been an active area of research. Several postharvest biocontrol products based on single antagonists have been developed and registered. The large scale commercial use of these products have been limited a due to inconsistent performance under commercial conditions . In this regard, Droby et al. (2018) have indicated that a new paradigm is needed for postharvest biocontrol to achieve commercial success and that understanding the naturally-occurring microbiome of fruit surfaces and its function, will lead to the development of new biological strategies for postharvest disease control. Several studies have reported on the population dynamics of biocontrol agents on intact and wounded fruit over the course of low-temperature storage. A wide array of mechanisms has also been demonstrated for postharvest biocontrol agents that involve yeast antagonist, the pathogen, and the host. This study and others are providing the foundation for understanding the structure and function of the carposphere microbiome. Such information is an essential step towards the development of effective biological approaches to postharvest disease management. For example, efforts to modulate the gut microbiome for improved human health have moved from simple inoculations with beneficial bacteria (probiotics) to supplements that contain specific metabolites that provide a resource that can be selectively utilized by beneficial bacteria (prebiotics) to combinations of probiotics and prebiotics (synbiotics) that can more effectively shift the composition of an existing host community . Similarly, in the apple rhizosphere, efforts to manipulate the soil microbiome to treat apple replant disease have shown that directed changes to the resource environment (e.g. through selective soil amendments) are more successful at controlling disease than inoculations with single strains or simple consortia of beneficial microbes [44–46]. Research designed to identify, quantify, and elucidate the metabolic networks constructed by microbial populations on harvested fruit is a fundamental need. Such studies will improve our understanding of the mechanisms that regulate the assembly of beneficial microbial communities, and lead to the development of strategies for beneficially manipulating microbial communities in situ.
Apples represent a major item of export and are shipped globally. Therefore, it is of importance to determine if the structure of the apple fruit microbiome is relatively uniform regardless of where the fruit is produced. Rather than the presence of a uniform microbiome, the present study revealed that geographical location is a principle factor determining the structure of the apple fruit microbiome. Fungal communities, however, were more affected by geographical location (country and site within a country) than bacterial communities. The stability of the fruit-associated bacterial community, relative to their fungal counterparts, has been previously reported in stored apples [11, 47]. The higher level of variation in the fungal community may be potentially attributed to the fact that fungal assemblages appear to be derived from regional fungal pools with limited dispersal capability . In addition, we observed that as the variation in the microbial communities among sites was positively correlated with the distance between those locations, especially for fungi. For example, variations in fungal and bacterial communities associated with apple fruit were lower at a local scale, i.e. among orchards within the same geographical location, sites within a country e.g. eastern and western USA and Canada and increased at the country level. Furthermore, a continental pattern can be drawn especially for the bacterial community which in one hand indicates adaptation of the apple microbiome to local environments, and on the other hand it may be explained by the metacommunity theory. A metacommunity is defined as a set of local communities that are linked by dispersal of multiple potentially interacting species . However, the present study had an insufficient distribution of samples to evaluate this premise. Nevertheless, the geographical location has been previously reported to be one of the most important determinants of the structure of the plant microbiome [50, 51]. A study of the maize rhizosphere found that location had a higher impact on the plant microbiome than genotype . Similarly, a study of the global citrus rhizosphere microbiome reported large variations in community structure that were attributed to geographical location (samples collected in different countries) . The large-scale variations between countries, together with the similarity observed among apple microbial communities within a country or region within a country, suggests that the structure of the microbial community on apple fruit is locally-adapted to local environmental conditions that influence microbial diversity and composition . In this regard, it is also commonly recognized that the humid, wet conditions present in the eastern portions of the USA and Canada, present a much greater disease and pest challenge than the dry conditions present in the western USA and Canada. This is especially supported by the differences in diversity levels between these two contrasting environments, although more evident for the fungal community (e.g. Figure 3).
Plants tissues provide a variety of niches that can harbor distinct microbial communities. Plant roots, leaves, flowers, fruit, as well as other organs, represent different microhabitats, each with specific features that favor the growth of specific microorganisms in these organs. Different tissue types within the same organ, have been previously reported to exhibit spatial variations in microbial community structure. For example, the upper and lower leaf sides, as well as the peel and pulp of various fruits, including apple, have been reported to exhibit differences in microbial community structure [11, 12, 55, 56]. The experimental design used in the present study was selected to determine if spatial variations within a fruit is global, i.e. will be evident regardless of geographical location and the variety of environmental conditions present in the different sites. Results indicated that the effect of fruit tissue-type on the composition of the microbial community was rather limited, R2 = 0.0266 for fungi and R2 = 0.05191 for bacteria, yet significant i.e. P = 0.001. A larger effect was observed, however, when individual orchards were analyzed separately (Figs. 5 and 6). Spatial variations in fungal and bacterial community composition and Shannon diversity due to tissue-type was consistently observed in all of the investigated orchards. These results, along with previous studies, confirms that spatial variation in the structure of the microbial community exist between tissue-types (calyx-end, stem-end. and peel) at a global level. Since geographical location, is the main factor shaping the structure of the apple microbiome, however, the effect of tissue-type is greatly reduced when samples of tissue-types are pooled across countries. Notably, the association of a distinct microbiome with such a small environmental niche (tissue-type) suggests specialized adaptation and function to those microhabitats. We suggest that the conditions (morphological, nutrient, and environmental) present in each of these microhabitats (tissue-types) could play an important role in determining community structure. For instance, the calyx-end is an open site that may create special niche for specialized fungi such as Alternaria and other fungal pathogens which can cause internal rots. Interestingly, Erwinia species were found at higher abundance in the Calyx-end tissue compared to the other tissue types, especially in Canadian apples. This can be explained by the fact that the calyx contains floral residues which are most affected by Erwinia amylvora, the cause of fire blight disease of pome fruit.
A core microbiome is a set of microbes consistently present over time on a specific host and is likely to be critical to host development, health, and functioning . Defining the core microbiome enables researchers to filter out transient associations and focus on stable taxa with a greater likelihood of influencing host phenotype and is therefore essential in exploring the potential for pre/probiotic treatments that support host health . In this study the core microbiome of apple fruit was defined as fungal and bacterial taxa present in at least 75% of all samples. We found two bacterial genera, namely Sphingomonas and Methylobacterium, and six fungal genera i.e. Aureobasidium, Cladosporium, Alternaria, Filobasidium, Vishniacozyma, and Sporobolomyces. This is a considerably low number of taxa, relative to other reported core microbiomes in plants . However, this can be attributed to the high number of samples in the present study; which lowers the probability that same taxon will be present in all samples and the evaluation of samples from different countries and tissue-types.
Sphingomonas, a gram-negative, non-motile, aerobic bacterial genus, is known for its bioremediation of heavy metals and biodegradation of polycyclic aromatic hydrocarbons, and is associated with plant growth promotion through its ability to produce gibberellins and indole acetic acid in response to different abiotic stress conditions, such as drought, salinity, and heavy metal stresses . Interestingly, those phytohormones are also involved in fruit maturation, development, and quality. For example, fruit-set in tomato (Solanum lycopersicum) depends on gibberellins and auxins [60, 61]. Similarly, Methylobacterium is a gram-negative, aerobic, motile bacterial genus with plant growth-promoting properties . Sphingomonas and Methylobacterium have been previously reported as a component of the apple microbiome and as two of their predominate genera [9–11, 47], as well as a component of the core microbiome in several other plant species [50, 63–65]. Aureobasidium and Cladosporium have also been reported as a common member of the microbiome of apple [12, 47] and other plants [66–68]. These taxa can be found as endophytes or epiphytes in association with various plant organs e.g. leaves, flowers, fruit, seed etc. Although the core microbiome is typically considered to have a high level of specificity between species, the common reporting of these taxa suggests the possibility of a core microbiome that is shared between different plant species. This commonality is expected to exist at the level of genus and that some degree of species specificity may exist. In this regard, we found that the core bacterial genera, Sphingomonas and Methylobacterium accounted for a considerable fraction of the observed variation between the investigated locations, as well as tissue types. For example, the community composition of either Sphingomonas and Methylobacterium was sufficient to distinguish between most of the investigated countries. Similar results were also observed for Aureobasidium, a core fungal genus, however, species variability was limited, and differences were attributed to niche specialization in the different tissue-types. Notably, both bacterial genera appeared to be distinct in tissue types. The geographical location demonstrated to be an important determinants of the Methylobacterium community composition in the plant phyllosphere . The presence of distinct Sphingomonas community in different fruit tissue-types suggests site-specialization to these microhabitats. Interestingly, the majority of the fungal core microbiome was represented by yeasts with known antagonistic activity against pre- and postharvest pathogens. Despite being one of the most common fungi associated with apples, Penicillium, the causal agent of the most important apple postharvest disease, blue mold [70–72], was not found to be a component of the core microbiome. Penicillium species are able to grow and proliferate at low temperatures during cold storage, giving them an advantage over other fungal species . In this regard and considering samples in the present study were collected immediately after harvest, it can explain the low prevalence and the absence of Penicillium from the apple core microbiome. Information about the core microbiome can be further used to develop biological control strategies against apple diseases and disorders. Though core species, by definition, are detected across all samples, their relative abundance pattern vary and, in some cases, forms characteristic groups of microorganisms. Dissecting the microbiome into co-occurrence modules can serve the construction of synthetic communities with distinct function . For example, such associations can serve the design of multiple-species synthetic communities for achieving an efficient biocontrol activity. Alternatively, dissecting the microbiome into microbial modules can allow limiting the search for a single efficient antagonist agent. In the context of the apple fruit microbiome, co-occurrence patterns have stratified the fruit microbiome into five key groups with core genera located in two of them: one cluster with Aureobasidium, and the second with Cladosporium, the two most abundant Ascomycota genera. Though most of the significant interactions detected in the network are positive, some negative associations allow formulating predictions for potential biocontrol agents against pathogens. Based on the network view, experimental design of potential biocontrol agent could compare the activity of a single microorganism vs consortium representing a native co-occurring module. Potential biocontrol strategies can hence benefit from the network view of microbiome interactions allow to go beyond the single biocontrol agent to the educated design of a biocontrol consortium.