Genotype-Dependent Recruitment of the Strawberry Holobiome

Cultivated strawberry (Fragaria × ananassa Duch., fam. Rosaceae) is an important fruit crop, greatly appreciated for its aroma and nutraceutical properties. Niche-specic characterisation of plant microbiome, from rhizosphere to aboveground plant organs, is crucial to understand the inuence of structure and function of the microbial communities on plant phenotype, performances and disease resistance. Strawberry cultivation is challenged by a large variety of pathogens, which cause substantial economic losses and require the frequent application of pesticides. Biological control is a promising and safer alternative to the use of xenobiotic pesticides. Biological control agents isolated from the microbiome of the host plant may have a superior ecacy in comparison to non-indigenous microbial inoculants. Therefore, the characterization of the native microbiome along different plant compartments is a key step for the successful microbial manipulation in farmlands. we provide parts

individual host and its associated microbial community 4,5 . The association between terrestrial plants and microbes developed at least 460 million years ago, as suggested by the fossil evidence of arbuscular mycorrhizae on some of the earliest land plants 4 . To date, many important questions regarding these associations remain unanswered, especially concerning the factors determining the community assemblage and diversity of the plant microbiome 3 . Increasing evidence suggests that plants can actively recruit a bene cial micro ora to facilitate their adaptation to environmental conditions and changes 3,6,7 . However, further studies are needed to generalize this hypothesis, and enable practical applications, especially for horticultural perennial crops grown in cultural conditions 8 . To date, most experiments on plant microbiome have focused either on speci c model plants (i.e. Arabidopsis thaliana) or economically important, annual herbaceous monocotyledons 9 . Perennial plants, on the other hand, are exposed to radically changing environmental conditions (including freezing winter temperatures, dry seasons, periodic ooding) 10 . Therefore, in perennial plants, the microbial community has evolved to last for more than a growing season, thus suggesting an assembly with a more intimate connection with host allowing its endurance to changing environmental conditions. Furthermore, perennial crops may promote plant-microbial linkages, increasing richness of bacterial and fungal bene cial communities, due to their extensive root networks and allocation of belowground carbon [11][12][13] . In addition, microbiome research has so far primarily taken into consideration the rhizosphere, while other plant compartments have been relatively neglected 14 . Finally, bacterial community analysis dominates the microbiome studies 15 . The study of bacterial and fungal microbiomes colonizing different plant compartments under agronomic conditions provides key information to unfold agricultural constraints and achieve a successful microbial manipulation in farmlands 16 .
The high adaptability of strawberry to different conditions, allows the cultivation under a wide range of environments and agronomical managements (from Mediterranean to the Nordic climates) making the fruit available on the market, almost independently of the season 19 . For this reason, strawberry fruit represents an important and valuable portion of the daily fresh food consumption 20 . Strawberry is greatly appreciated for its aroma and nutraceutical properties. Among others, strawberry fruit contains phytochemicals, such as anthocyanins and ellagitannins which may prevent human health diseases induced by reactive oxygen species 21 . While strawberry productivity and quality can be positively improved by bene cial microorganisms 22 , the cultivation is challenged by a large variety of pathogens, which cause substantial economic losses and require the frequent application of pesticides. Among these diseases, red stele (Phytophthora fragariae), powdery mildew (Podosphaera aphanis) and leaf spot are the ones most severely affecting strawberry production worldwide 23 . Powdery mildew mainly affects photosynthetic ability of strawberries cultivated in humid environments 24 , which leads to strong reduction of growth and productivity with major yield losses 25 . Leaf spot diseases, which in severe conditions may led to plant death, are caused by different pathogens, including bacteria (Xanthomonas fragariae) and fungi (Colletotrichum gloeosporiodes, Mycosphaerella fragariae, Cercospora fragariae, Mycosphaerella louisianae, Septoria fragariae, S. aciculosa, S. fragariaecola, etc.). Multiple resistance to a broad spectrum of diseases such as powdery mildew and leaf spot is still not available among commercial strawberry cultivars (i.e. human-selected clonal genotypes) 26 . Disease control is particularly challenging in strawberry production, since several cultivars present at the same time, owers, fruit and leaves, and are therefore subjected to a high risk of pesticide residue accumulation on berries 19 . Biological control is a promising and safer alternative to the use of xenobiotic pesticides. Some commercially available, bene cial microorganisms (i.e. Ampelomyces quisqualis, Bacillus subtilis, Trichoderma harzianum, Glomus spp.) have been tested for disease control in strawberry, yet none of them has demonstrated characteristics of reliability, persistence and/or cost-effectiveness justifying their use as an alternative to chemical pesticides 23 . The unsatisfactory degree of disease control and the high variability of results obtained in different locations and seasons with commercial bene cial microorganisms can be explained by the fact that those microbes are in most cases non-native to the strawberry plant microbiome. Several studies suggest that biological control agents isolated from the microbiome of the host plant have a superior e cacy in comparison to non-indigenous microbial inoculants [27][28][29] . Thus, the characterization of the native microbiome is a key step for the successful selection of bene cial microorganisms against plant diseases 1 . Unfortunately, the complete microbiome of cultivated strawberry has not yet been described, hindering the identi cation and selection of the most effective indigenous microorganisms to improve plant tness and fruit quality and/or provide resistance to biotic and abiotic stresses.
The aim of this study was to provide a complete picture of the strawberry holobiome, including both fungal and bacterial populations, and to identify a core microbiome, from soil, plant-soil interface (rhizosphere) and plant compartments (roots and above-ground organs) using Next Generation Sequencing (NGS). For this purpose, three commercially important strawberry genotypes ('Elsanta', 'Darselect' and 'Monterey') were used. Furthermore, the effects of strawberry genotypes, soil and plant compartments on the richness and community composition of the overall microbiome were studied, with a focus on pathogenic and bene cial microbes. Finally, the links between strawberry microbiomes, plant mineral nutrient content and fruit quality traits were investigated. To our knowledge, this study provides the rst in depth and comprehensive view of horticultural crop microbiome in relation to plant genotype, health and nutritional status and fruit quality parameters, shedding light on potential practical applications to increase the sustainability of crop production.

Results And Discussion
Composition of strawberry microbiomes Quadruplicate bulk soil, rhizosphere, root and above-ground organs samples were prepared for bacterial 16S rRNA and ITS gene community pro ling for three strawberry genotypes (Fig. 1). In roots and aboveground organs, we targeted epiphytic and endophytic microorganisms jointly. In total, we generated 1,531,637 (average of 31,909 reads per sample) and 739,458 (average of 15,405 reads per sample) high quality reads excluding chimeric sequences for bacteria and fungi, respectively. We removed singletons which may come from sequencing errors and normalized all bacterial and fungal datasets to 10,930 sequences for bacteria and 8,077 for fungi. Rarefaction curves show the su cient sequencing effort for most of the samples (Fig. S1b,c). Nevertheless, OTU richness estimates, predicted with Chao1 were also analysed and showed (Fig. S2). We used observed richness directly as diversity measure for both bacteria and fungi (Fig. 1c,e). In total, we detected 26,434 bacterial and 1,716 fungal OTUs. The total bacterial and fungal community assemblages were compared using two-way PERMANOVA to identify the main drivers of the microbiome composition ( Table 1; Table S1). Notably, we found that microbial compositions are strongly dependent both on the analysed genotype (bacteria F = 1.  Table 1; Table S1). Similar results were obtained when we compared the effect either of genotype or compartment by means of twoway ANOSIM analysis (Table 1; Table S1). In agreement with previous studies 4,30 , above-ground organs displayed the lowest bacterial and fungal OTU richness. 'Darselect' showed to be the genotype with the lowest bacterial richness in all compartments (Fig. 1c). OTU richness estimates, predicted with Chao1, also showed similar results as the observed data (Fig. S2). Diversity between above and below-ground microbial community composition ( Fig. 1a; g. S1a) and richness could be explained by the differences in the physical and chemical properties of the two environments. In fact, above-ground organs are subjected to oligotrophic and unstable conditions (with daily and seasonal uctuations in temperature, humidity, UV light 31 ), whereas the soil compartment is relatively more protected, stable and nutrient-rich 32 .
Our study showed that Actinobacteria, Alphaproteobacteria, Gammaproteobacteria, Deltaproteobacteria and Bacteroidia are the bacterial groups representing the backbone of strawberry bacterial microbiome in all plant and soil compartments, all together accounting on average for 80% of total detected OTUs based on presence-absence data (Fig. 1a), which correspond to 93% of total bacterial abundance (Fig. S1a). Above-ground organs of 'Darselect' and 'Elsanta' were dominated by Actinobacteria (54 and 53% respectively), whereas 'Monterey' cv was mosty colonized by Gammaproteobacteria (Fig. 1a). Belowground compartments of the three cvs were dominated by Alphaproteobacteria (Fig. 1a). Analyzing bacterial abundance, we found that Actinobacteria was the predominant group in all genotypes and compartments analysed. The lowest percentages were found in the rhizosphere and bulk soil of 'Elsanta' (31 and 33%, respectively), whereas 'Elsanta' and 'Darselect' above-ground compartments showed a high group homogeneity, being dominated by Actinobacteria for 98 and 99%, respectively (Fig. S1a). Alphaproteobacteria were homogenously represented in plant and soil compartments of the three genotypes. Gammaproteobacteria were almost absent in above ground compartments of 'Elsanta' and 'Darselect', while they were the second most represented group in 'Monterey' (26%) (Fig. S1a).

Identi cation Of The Core Microbiome
In this study, we report the existence of a core microbiome, common to the three genotypes (Fig. 2). The dominant bacterial and fungal groups in the overall and core microbiome are similar, at the ne taxonomic resolution. We identi ed ubiquitous microbes in all the studied environments from the soil to the above ground plant organs of all three strawberry genotypes. This observation suggests that they are either able to colonize all soil and plant compartments or they can move across soil and the plant organs with a passive or active translocation from roots to the above-ground organs (e.g. leaves, runners).
Among these core microbes, 24 OTUs were bacterial (mainly Micrococcales) and 15 fungal (mainly Ascomycota). Interestingly, several strawberry pathogens were found among the core fungal OTUs, namely Plectosphaerella cucumerina (fruit, root and collar rot), Botrytis caroliniana (gray mold) and Alternaria alternata (black leaf spot) ( Table S2). The colonisation of plant organs by a pathogen is not su cient, per se, to result in a successful infection causing disease symptoms 33 . This observation con rms that abiotic (e.g. temperature, humidity, nutrient availability) 34 and biotic (e.g. plant-associated microbial consortia, plant resistance) 23,35,36 factors play a crucial role in determining the fate of plantpathogen interactions.

Functions Potentially Expressed By The Microbiota
Based on their taxonomy, 3,845 bacterial (15% of all detected bacteria) and 706 fungal (41% of all detected fungi) OTUs were assigned to a putative functional group. Results showed that 20 bacterial and 16 fungal functional groups colonized different soil and plant compartments of strawberry plants (Table  S3). Chemoheterotrophy, methanol oxidation, intracellular parasitism, predation/exoparasitism were the dominant bacterial functions while saprophytism, plant pathogenic and endophytic colonization were dominant among the fungal functions (Table S3). Within the bacterial OTUs we further explored speci c functions relevant to plant health, tness and growth. We identi ed 285 OTUs as potential N-xing genera and 129 OTUs as species known for their activity as biological control agents (BCA) and/or plant growth promoter (PGPB) ( Fig. 2; Fig. 3b; Table S4). Interestingly, both compartment and genotype had a signi cant role in de ning plant-associated bene cial bacterial community, according to ANOSIM and PERMANOVA (P < 0.001) ( Fig. S3a; Table 1; Table S1).
It has been suggested that domestication of crop plants has determined a reduction in the biodiversity of the associated micro ora, in particular for functions regarding nutrition and stress tolerance 37 . On the other hand, it is also possible that cultivated plants recruit microbes speci cally exerting bene cial functions under cultural conditions. In this view, the ability to interact with such microbes may be regarded as a trait selected by domestication 38 . In this work, we found that, even after centuries of domestication and complex hybridisation 17,39 , cultivated strawberry plants are associated with 16 nitrogen xing bacterial genera (Fig. 3a), which is more than what reported in wild strawberry plants relatives (F. chiloensis, F. virginiana ssp. platypetala, F. × ananassa ssp. cuneifolia) (7 genera) 40 and comparable to the number of nitrogen-xing genera (18) reported in legumes, which are nodulating plants specialized for symbiosis with nitrogen-xing bacteria (Table S6). The presence of nitrogen xing bacteria is con rmed by PCR on nifH gene in bulk soil, rhizosphere and root samples of the three strawberry genotypes (Fig. S4).
Although, bacterial taxa known to have N-xing potential were surprisingly found in the above-ground habitat (Fig. 3a), we did not detect any nifH gene in this compartment (Fig. S4). Indeed, nitrogenase is inactivated by oxygen. This may indicate that the ability of these bacteria to interact with plant hosts is at least partially disconnected from the ability to x nitrogen.
Our work also revealed the vast diversity of fungal partners of strawberry, which have not been thoroughly investigated so far, and include ectomycorrhizae, arbuscular mycorrhizae, ericoid mycorrhizae, endophytes, dark septate endophytes and mycoparasites (Figs. 2 and 3c,d ).
Remarkably, we found that both genotype (F = 3.34, P = 0.001) and plant compartment (F = 3.90, P = 0.001), as well as their interaction (PERMANOVA values genotype × compartment F = 1.58; P = 0.002; Fig. 4b; Fig. 3b; Table 1; Table S1) play a key role in the abundance of pathogens in the fungal community associated to strawberry.
The environmental factors, soil conditions and pool of natural microbial inoculum are assumed to be comparable for all three strawberry genotypes, as plants were grown in the same cultural and environmental conditions. Therefore, the observed differences in associated bacterial and fungal communities (Figs. 2, 3) can be explained with the ability of the plant to adjust the composition of the associated micro ora 41 . In this view, the lower susceptibility to powdery mildew and leaf spot observed in 'Monterey' over the season (Table S8), may be at least partly due to its ability to establish exclusive bene cial microbial relationships (Fig. 4a,c).
Indeed, while most of the potentially bene cial fungal groups are similarly represented in the three strawberry genotypes, the arbuscular mycorrhizae Rhizophagus irregularis showed a high frequency only in 'Monterey', while being completely absent in 'Elsanta' and 'Darselect' (Fig. 3d). In several crop plants, the colonisation of the root systems by R. irregularis has been demonstrated to confer plant resistance to broad-spectrum of pathogens by induced systemic resistance (ISR) and mycorrhizal-induced resistance (MIR) 42,43 . Regarding the bacterial bene cial microbiome, in cultivar 'Monterey', 19% of bene cial OTUs were able to simultaneously colonise below and above-ground organs, whereas in Elsanta and Darselect only one OTU (identi ed as B. megaterium) was found to colonize both underground and above-ground organs. B. megaterium has attracted considerable attention as a functional microbe in several crop species, including strawberry, since it is able to solubilize phosphate and produce phytohormones 44 . Furthermore, it has been proven to be effective for the control of B. cinerea 45 .
Surprisingly, Pseudomonas uorescens has been detected only in the above-ground compartments of Monterey , the genotype showing the highest disease tolerance (Fig. 3b). We further investigated its colonization ability by PCR ampli cation. Indeed, we proved the ability of Ps. uorescens to establish detectable populations in the soil and, remarkably, both in internal and external tissues of Monterey plants (Fig. S5). Many Ps. uorescens strains have been proven to promote plant growth or protection, by mechanisms such as phosphorus solubilization, phytohormone production, competition against phytopathogens, elicitation of ISR, or production of antimicrobial compounds, such as cyanide or phenolics 46,47 . Non-indigenous Ps. uorescens strains have been already applied to strawberry plants, allowing to anticipate owering and fruiting, increase fruit yield and vitamin content 48 , and to control crown rot (Phytophtora cactorum) 49 . Notably, the inoculation of rice seed with a Ps. uorescent strain for riceblast control resulted in the colonization of roots, stems and leaves 50 , supporting that this species does not have strict organ preferences.
Interestingly, the combined action of Pseudomonas spp. and Rhizophagus spp. has been explored in several crop species 43,51,52 . In particular, a mixture of AMF, which included Rhizophagus sp., and Pseudomonas uorescens was successfully applied to strawberry, resulting in increased fruit production and quality 48 . The combination of Rhizophagus sp. and Ps. uorescens has been proven to elicit plant systemic defence system in tomato via the activation of ethylene response to pathogen attack 43 .
Finding unique bene cial microbial patterns for a genotype that showed to be more tolerant than others to biotic stresses suggests an important contribution of the microbiota in the defence strategy of strawberry plants. In uence of rhizosphere microbiome on plant tolerance to root diseases is well known 53 . However, microbiome investigations focusing on speci c soil or plant compartments may be less informative than studying the overall plant holobiont. In our work, we show a clear relationship between plant tolerance to above-ground diseases and overall plant colonization by speci c microbes.
Howbeit, further studies are required to deeply investigate, and nally agronomically exploit, the naturally occurring, genotype-speci c plant bene cial microbiome.
Interactions between strawberry microbiome, plant mineral composition and effect of microbiome on fruit quality Besides nding signi cant effects of genotypes, soil and plant compartments on the taxonomic ( Fig. 1b,d) and functional composition of both bacterial and fungal communities (Figs. 4b; S3, Table 1), we found signi cant correlations between the mineral composition of the plant organs and the microbial community assemblage of bacteria and fungi across the different soil and plant compartments (Fig. 5a). Indeed, plant associated microbiomes have been already proven to play a key role in improving plant nutrition both by promoting nutrient acquisition and nutrient use e ciency 54 . On the other side, the host plant and its nutrient preferences impact its microbiome recruitment 55 .
In addition, microbes, and particularly those associated with soil and roots, contribute substantially, although indirectly to sensorial fruit quality (Table S11). In details, titratable acidity is mainly related to the below-ground microbiome (Fig. 5b), whereas total soluble solids content of fruits is linked to rhizospheric and above-ground bacterial microbiome and to below-ground fungal microbiome. Notably, inoculation of Bacillus sp. on owers and leaves of sour cherry affected sugar content and titrable acidity of fruits 56 . Similar results were obtained applying bacteria and AMF, both alone or in combination, on strawberry plantlets 22 . Fruiting process and ripening are nely regulated by phytohormones, in particular by ethylene, auxin and gibberellins that are known to be produced by both fungi and bacteria. Ethylene is a key regulator of fruit ripening thus in uencing all the main quality traits. Despite strawberry has been considered as non-climacteric fruit, new genetic evidences suggest that ethylene is required for strawberry ripening 57,58 . Ethylene is produced by a wide range of microbes starting from two alternative precursors, 2-keto-4-methyl-thiobutyric acid (KMBA) or 2-oxoglutarate 59,60 . Furthermore, several bacterial species present the 1-aminocyclopropane-1-carboxylic acid (ACC) deaminase activity. ACC deaminase degrades the ethylene precursor, thus, impairing its production in the plant tissues 8 .
AMF have been proven to affect plant hormonal balance and metabolism, indeed their bene cial effect has been observed both in below and above-ground organs 48 . Besides AMF, PGPB are also able to affect fruit quality, mainly by modulating the interplay between ethylene and auxin metabolisms and proving essential nutrients 8,61 . Altogether, these correlations suggest that bacteria and fungi contribute to the host's adaptation to growing conditions and, consequently, to fruit development.

Conclusions
Cultivated strawberry genotypes interact with a variety of microbial species. Such interactions have been demonstrated to be speci c to genotypes and compartments. These microbiomes play a key role in the plant ability to cope with biotic stress and in modulating fruit quality. Our ndings suggest that a comprehensive picture of plant holobiome is needed in order to shed light on the in uence of microbial communities and key microbes on plant phenotype and performances. Further studies on microbiomes of crop plants can contribute to the advancement of plant production science, by providing a deeper insight in the interactions between crops and the micro ora and evidencing applicative tools and strategies for an e cient and environmentally sustainable horticultural practice. However, the complexity and speci city of the patterns described in this work suggests that the idea to replace agro-chemicals by a few universal bene cial microorganisms is not realistic. Therefore, breeding programs should aim at the selection of high quality, climate-change resilient horticultural varieties with remarkable capacity to establish symbiotic relationships with useful microorganisms 8  11°22'E, 450 a.s.l.). Plants were fertigated using a drip system (Table S9). Throughout the season, addition 100 plants of each genotype grown in same conditions were weekly monitored for powdery mildew and leaf spot symptoms. Symptom severity on leaves was visually ranked using a 0-5 scale (0 = no symptoms; 5 = plant death) (Table S7) The presence of nifH gene in samples was veri ed by PCR using nifH gene-speci c primers (PolF (5'-TGC GAY CCS AAR GCB GAC TC -3') /PolR (5'-ATS GCC ATC ATY TCR CCG GA -3')), as previously described 64 .
Pseduomonas uorescens detection in 'Monterey' genotype was performed as follows: bulksoil and rhizosphere DNA were extracted as above; roots and above ground parts of strawberry plants were surface sterilized two times with deionized water and 70% ethanol and washed 3 times sterile water, organs were let 3 h in sterile water. DNA was extracted as above and ampli ed using Pseudomonas uorescens speci c primers (16SPSE uF (5'-TGC ATT CAA AAC TGA CTG-3') /16SPSER (5'-AAT CAC ACC GTG GTA ACC G-3')) as described elsewhere 65 . Both for nifH and P. uorescens, ampli cation products were visualized through agarose gel 1.5% electrophoresis.

Analysis Of Plant Mineral Composition And Fruit Quality Traits
Ultrapure 65% HNO 3 was obtained from analytical grade HNO 3 (Table S10).
Strawberry fruit rmness was measured by a texture analyser (Zwick Roell, Italy) using the penetration test methodology that was previously developed for raspberry 66 . This penetration test outlined a mechanical force displacement using a 5 kg loading cell and a cylindrical at head probe with a diameter of 4 mm entering into the berry esh that was placed on the plate with the receptacle upright to the compression probe. Mechanical pro les were acquired with a resolution of 100 points per second with the following instrumental settings: test speed of 300 mm min − 1 , post-test speed of 1000 mm min − 1 , auto force trigger of 2 g and stop plot at target position. Each berry was penetrated until a 99% penetration strain. In this study only the maximum force value (N) was considered, since this parameter is usually highly related with berry rmness 66 .
Soluble sugar content was measured on strawberry fruit juice with a hand-held Atago digital refractometer (Optolab, Modena, Italy). Titratable acidity was determined on strawberry juice diluted (1:2) in distilled water by titration with NaOH to pH 8.1, and expressed as citric acid equivalents.

Bioinformatics
High quality reads from the paired-end sequences generated by Illumina MiSeq sequencing platform were extracted using MOTHUR 67 and OBI Tools 68 software suits. PANDAseq was used to merger forward and reverse raw reads from the same sample by using the simple-bayesian algorithm with a minimum overlap of 80 and 20 nucleotides for bacteria and fungi, respectively. All the merged reads were then trimmed with the following parameters: (i) minimum length of 350 (bacteria) and 120 (fungi), (ii) minimum average Phred score of 25 on the trimmed length, (iii) no ambiguities in the sequence length, and (iv) maximum length of 20 homoploymers in the sequence. The reads were then pre-clustered using CD-HIT-EST, allowing a maximum of 1% of dissimilarity and with only one base allowed per indel 69 , in order to merge those reads arising likely from sequencing errors 70 . Chimeric sequences were detected using the UCHIME algorithm 71 as implemented in MOTHUR and removed. Reads from each sample were pooled together and were dereplicated into unique sequences and sorted by decreasing abundance. The resulting reads were then clustered into operational taxonomic units (OTUs) using the CD-HIT-EST algorithm 72

Statistical analysis:
To assess the coverage of the sequencing depths, individual rarefaction analysis was performed for each sample using the function 'diversity' in PAST. At the analyzed sequencing depths, all individual rarefactions shown to be su cient to infer bacterial and fungal community composition and richness in our samples (Fig. S1). We de ned core microbiome as the bacterial and fungal communities that are comprised of OTUs that were detected in all strawberry genotype and present in more than 75% of the samples 78 . The effects of strawberry genotype, soil plant compartment (bulk soil, rhizosphere, root and aboveground organs) on bacterial and fungal OTUs richness were analyzed using two-way analysis of variance (ANOVA), incorporating the Jarque-Bera JB test for normality. The effects of strawberry genotype, soil and plant compartment on bacterial and fungal community compositions were visualized using Non-metric multidimensional scaling (NMDS) based on the presence-absence data and Jaccard distance measure. Coloured ellipses in NMDS ordinations are 95% con dence intervals of species centroids for each treatment level. The signi cant effect of the strawberry genotype, soil and plant compartment on bacterial and fungal community compositions were determined using two-way Analysis of Similarity (ANOSIM) and two-way Permutational multivariate analysis of variance (PERMANOVA) based on the presence-absence data and Jaccard distance measure over 999 permutations. Since relative abundance data from Next Generation Sequencing may not be fully used quantitatively 79 , we analyzed the microbial community composition using both presence/absence and relative abundance data sets. The results from presence/absence data are presented in the main text and the corresponding results using relative abundance data (with Bray-Curtis distance measure) are presented in Supplementary Information (Table S1). NMDS ordination based on presence/absence data and the Jaccard dissimilarity measure coupled with the env t function of the vegan package in R were used to investigate the links between each of bacterial and fungal community composition (bulk soil, rhizosphere, root and aboveground organs) and soil nutrient parameters, strawberry genotypes, fruit quality parameters (soluble sugar content and titratable acidity). NMDS stress values were between 0.06-0.13.
All statistical analyses were performed using PAST 80  Authors contribution: FS and WP conceived project idea and designed the experiments. BF and ID carried out the eld experiment. EF analyzed the mineral composition. WP, BT, DoS, ID and DS performed molecular analysis. BF, ID and AC performed the biochemical analysis. ID performed the classical microbiological and fruit quality analysis. SFW contributed for bioinformatics. WP, DS and BF contributed for statistical and data analysis. AC, DS, ID, EF, BT, SFW, DoS, BF, FB, FS and WP contributed to critical discussion and revision of nal manuscript. All authors read and approved the nal manuscript.