Positive response of barley to an indigenous arbuscular mycorrhizal fungal (AMF) inoculant is modulated by genotype and environment through changes in AMF root abundance and community structure

Positive response of barley to an indigenous arbuscular mycorrhizal fungal (AMF) inoculant is modulated by genotype and environment through changes in AMF root abundance and community structure | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Positive response of barley to an indigenous arbuscular mycorrhizal fungal (AMF) inoculant is modulated by genotype and environment through changes in AMF root abundance and community structure Valentina Marrassini, Laura Ercoli, Ana Vanessa Aguilar Paredes, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4314201/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 5 You are reading this latest preprint version Abstract In barley cultivation, high use of mineral fertilisers in combination with low crop nutrient use efficiency results in severe environmental and economic issues. In this context, inoculants with indigenous arbuscular mycorrhizal fungi (AMF) could represent an efficient solution where intensive agriculture negatively impacted soil AM fungal abundance and diversity. However, since crop breeding and environment can strongly affect plant mycorrhizal response, in this work, we tested the agro-ecological effect of field inoculation with a indifìgenous AM fungal consortium on three varieties of barley for two years. In 2020, when soil was clay loam with very low P availability and no drought stress, Atomo and Concerto varieties positively responded to inoculation in terms of AM fungal traits, whereas in 2021, with silty clay loam soil, low P availability and drough stress, only Concerto was responsive. In 2020, inoculation promoted grain yield by 64% and 37% in Atomo and Concerto, and in 2021 by 78% and 134% in Concerto and Atlante. Multivariate analysis highlighted a strong effect of environment on barley productivity and a third-order significant interaction AMF, genotype and environment (65% and 7% of explained variance). Inoculation slightly modified AM fungal composition, it strongly modified, together with plant growth stage, the AM fungal community structures. A significant relationship between root AM fungal abundance and barley productivity was highlighted, with arbuscules as best predictor. Accordingly, changes in AM fungal root community structure and not in composition drove barley response and the main players were Glomus sp. VTX00342 and Septoglomus sp. VTX00064, putative members of the local AM inoculum. The general positive barley productivity outcome supports the use of indigenous AMF for building efficient and ecologically safe inoculants and their inclusion in sustainable agriculture. Nevertheless, the selection of genotypes with stable AM fungal response in specific climatic conditions is crucial in biofertilization programmes. native arbuscular mycorrhizal fungi barley genotypes barley nutrient uptake mycorrhizal yield benefit molecular diversity field inoculation Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 1. Introduction Barley ( Hordeum vulgare L.) production had increased steadily for over 30 years, but it should improve by 50% or even more by 2050 to ensure global food security (Fischer et al. 2014). However, the high intensity and frequency of extreme events due to climate change has severely affected crop production (Mall et al. 2017; Meza et al. 2020; Trenberth et al. 2014). Drought is reported to cause strong reductions of barley biomass and yield (Guo et al., 2009; Ozturk et al., 2002; Rollins et al 2013; Sallam et al., 2019; Samarah 2005). In addition, a decline of mineral concentration in grain under rising CO 2 was highlithed (Gojon et al. 2022). Moreover, high use of mineral fertilisers in combination with low crop nutrient use efficiency results in a severe environmental issue and a high economic weight for farmers (Chien et al., 2009; Mclaughlin et al., 1996). These issues can be addressed either through the implementation of management practices (Mhlanga et al., 2021; Pittelkow et al., 2015; Sharma et al., 2021), or by the plant breeding (Lammerts van Bueren and Struik, 2017). In such a context, microbial biostimulants could represent an efficient tool to support barley productivity, especially under abiotic stresses, and to reduce the use of mineral fertilisers (Larimer et al., 2010; Philippot et al., 2013; Schütz et al., 2018; Veresoglou and Menexes, 2010). Among beneficial microbes, arbuscular mycorrhizal fungi (AMF) (Glomeromycota; Tedersoo et al. 2018) has been reported to improve in field conditions wheat grain yield by 20% (Pellegrino et al 2015). This was further confirmed by Zhang et al. (2019) who estimated an increase of 16%. Mixed-species AM fungal inocula determined higher grain yield than single species. The highest AM fungal contribution to grain yield was detected at low soil N and P availabilities and in sandy soils. Wheat grain and straw P concentration were promoted through field AM fungal inoculation by 7% and 19%, respectively, while grain N content and Zn concentration by 31% and 7% (Pellegrino et al., 2015). Barley is considered to be non responsive to AMF (Grace et al. 2008). Nevertheless, colonization under controlled and field conditions has been consistently reported (e.g., Clarke and Mosse, 1981; Jensen 1982; Masrahi et al. 2023; Powell et al. 1980). A neutral effect of AMF on yield was reported by Zhang et al. (2019), but the authours based this result only on three experiments and highlighted the need for improving data collection. In field conditions, inoculation with Gigaspora margarita promoted plant growth of barley by 92% (Powell et al. 1980). Under low P soil availability, ear fresh weight was doubled, irrespective of inoculated AM fungal species (Clarke and Mosse, 1981). Inoculants composed by three exotic AM fungal species increased grain yield and P uptake by 27% and 35%, respectively, while no effect was reported with native AM fungal mixtures (Powell et al., 1981). By contrast, no changes were reported on grain yield and P uptake of barley inoculated with Funneliformis mosseae (Khaliq and Sanders 2000). Field inoculation with F. mosseae in not fumigated plots resulted in significant depressions of grain and straw yield (-20%) (Khaliq and Sanders 1998). Therefore, under field conditions the outcome of the AM fungal inoculation was reported to be variable. This can be due the several factors, such as the abundance of infective propagules in soil, plant-fungal compatibility, composition of the AM fungal inocula, availability of soil nutrients and climatic parameters (Verbruggen et al., 2012). Intensive agriculture practices, such as high P or N fertilizer rate, ploughing and continuous monoculture, have been shown to negatively impact the abundance, diversity and functionality of AMF in soil (Verbruggen et al. 2010a,b). Indeed, land use intensification modified the composition of AM fungal communities which resulted dominated by few taxa within Glomerales with low crop functionality (Oehl et al. 2010; Pellegrino et al., 2014). Therefore, it clear that in soils with low biological fertility is important to improve the abundance and diversity of AMF through inoculation in order to maximize their benefits on crops (Yang et al. 2014). The variability in plant benefits to AMF was explained by the identity of plant host and fungus (Klironomos 2003; Maherali and Klironomos 2007; Mensah et al. 2015; Munkvold et al. 2004). Large differences were observed among genotypes/cultivars of wheat (Hetrick et al. 1993, 1996). By contrast, barley was less investigated. Al Mutairi et al. (2020), testing the effect of inoculation of R. irregularis on five barley cultivars, observed that genotype was a very strong driver for biomass, yield and yield components. Therefore, the study of barley intraspecific response to AMF it is of current great importance. Moreover, since the interaction between cereals and AMF was modulated by environmental factors (Grey 1991; Jerbi et al. 2020; Marrassini et al. 2024; Pellegrino et al. 2015), there is the need to carry out multiyear field studies. Commonly, commercial AMF inocula are composed of generalist single or few exotic AM fungal species, having low genetic variability and not always offering efficiency and stability when applied (Salomon et al. 2022). Some commercial inoculants failed to form mycorrhizal associations, which may be caused by low adaptation to local edaphic conditions (Schreiner 2007). Moreover, since AMF were generally considered mutualistic, there has been little concern over potential negative consequences of their introduction. Nevertheless, the evidence that mycorrhizal function can range from mutualistic to parasitic (Johnson et al. 1997; Jones and Smith 2004; Klironomos 2003) led to take into account the potential agroecological concerns of exotic AM fungal introduction (Schwartz et al. 2006). Several studies were successfully carried out in the field with indigenous AMF, inoculated as single or mixture, on many field crops, and indigenous AMF were often reported to be more beneficial and less agroecologically harmful than exotic strains (Jansa et al. 2008; Oliveira et al. 2005; Pellegrino et al. 2011; Pellegrino and Bedini 2014). Therefore, in this work, we inoculated a indigenous AM fungal consortium on three barley varieties for two years of cultivation to dissect the effect of the interaction AMF, genotype and environment on barley productivity, removing the effect of potential changes in AM fungal composition due to inoculation with exotic AMF (Fig. 1). In the present work, the benefit of AM fungal inoculation was evaluated by assessing grain yield and nutrient concentration (Fig. S1). Abundance and the community composition and structure of AMF in roots were evaluated using morphological and molecular tools. The indigenous inoculum was composed many fungal species, isolated from soil located in the same agricultural area where the experiment was carried out. We tested the following hypothesis: (i) barley genotype exerts a greater control over the response of the plant to AMF than the environment; (ii) under inoculation with indigenous AMF, host plant preference in AMF colonizing plant genotypes is driven by changes of community structure and not by changes in the composition; (iii) plant growth stage has a role in modulating the preference of barley genotypes; (iv) increases in AM fungal abundance and changes in community structure and not in composition determine barley productivity. 2. Material and Methods (2019 vs 1463 in altro MS) 2.1 Fungal and plant material The AM fungi used as inoculant were a consortium of taxa originating from a local field (Pellegrino and Bedini, 2014). The AM fungal inoculant was composed of 14 species belonging to five families: Acaulospora cavernata , Acaulospora spinosa , Acaulospora spp., Diversispora spurca , Funneliformis coronatum , Entrophospora etunicata (syn. Claroideoglomus etunicatum ), Funneliformis geosporum , Funneliformis mosseae , Glomus spp., Rhizophagus clarus , Rhizophagus irregularis , Scutellospora aurigloba , Scutellospora calospora and Septoglomus viscosum . Three barley varieties were tested: Atlante (six-row, intermediate growth habit), Atomo (two-row, winter growth habit), and Concerto (two-row, spring growth habit) (Limagrain Italia SpA, Fidenza, Parma, Italy). For details about barley varieties see Table S1. 2.2 Experimental field site The experiment was carried out in 2020 and 2021 at the ‘Società Cooperativa Rinnovamento Agricolo’, Santa Luce, Pisa, Tuscany (43 ° 26’24’’N-10 ° 29’48’’E; 37 m above sea level) in two adjacent fields. The soil of 2020 and 2021 showed difference in texture (clay loam and silty clay loam, respectively), but similar low nutrient availability (Table S2 and Supplementary Material and methods 1). The climate of the site is cold and humid Mediterranean (Csa), according to the Köppen-Geiger climate classification (Kottek et al. 2006) with a five-year average annual precipitations of 1033 mm, a 10-year average of annual maximum and minimum daily air temperature of 20.4 °C and 10.8 °C, respectively. During barley cropping cycle in 2020 (January-July), mean maximum and minimum temperatures and total precipitation were 20.4 °C, 10.2 °C and 373 mm, while in 2021 (March-July) 22.7 °C, 11.7 °C and 170 mm, respectively (Fig. 2). 2.3 Experimental set-up and sampling A complete factorial experimental design with three factors was adopted with two years of cultivation (2020 and 2021), three barley varieties, and two AM fungal inoculation treatments (inoculated with the AM fungal consortium, +M; not-inoculated/control, -M) (Fig. 1). The experiment was arranged in a completely randomized design with three replicate plots (8 m x 42 m = 336 m 2 in 2020; 8 m x 20 m = 160 m 2 in 2021). The inoculum, produced as described by Pellegrino and Bedini (2014), was a micronized mixture of mycorrhized roots of sorghum ( Sorghum halepense L.), spores, hyphal fragments, and bentonite as carrier. The inoculum was distributed at the sowing by manual application to seeds that had been previously moistened with water. The rate of the AM fungal inoculum was 0.8 g m 2 (8 kg ha -1 ) (1.2 kg of inoculum 100 kg -1 seeds, about 2500 spores ha -1 ). The mock inoculum (not-inoculated/control) consisted of the same dose of steam-sterilized AM fungal inoculum (121 °C for 25 min on two consecutive days). To ensure a common microflora, both inocula received 0.05 L kg -1 of a filtrate obtained by filtering through a Whatman no. 1 filter paper the AM fungal consortium. The seed rate was 200 kg ha -1 , corresponding to approximately 350 viable seeds per m 2 , distributed in rows 14 cm apart. Barley was seeded with a pneumatic seeding machine (Aguirre Bota) on 17 January 2020 and 1 March 2021. Before the experimental setup, the preceding crop was clover ( Trifolium alexandrinum L.) in 2020 and faba bean ( Vicia faba L. var. minor) in 2021. Soil tillage was carried out in autumn by moldboard plowing at a soil depth of 30 cm, and by harrowing at a soil depth of 15 cm, immediately before seeding. No organic/chemical fertilizer was applied. No weed and pest/pathogen control treatments were applied. In both years of cultivation at the stage of four leaves unfolded (GS14) (Zadoks et al. 1974) and at the physiological maturity (GS90), ten plants, randomly selected in each replicate plot, were excavated with their root system to determine AM fungal abundance and diversity. Barley was harvested on 22 July 2020 and 26 July 2021 in each replicate plot by a combine harvester (Laverda, Vincenza, Italy). Furthermore, in 2021, at GS14, shoots from the ten plants were also sampled. 2.4 Mycorrhizal abundance in barley roots At each sampling, fresh roots from each replicate plot were combined and cleaned from the attached soil by soft washing with tap water. Mycorrhizal abundance was measured by the percentage of root length containing arbuscules and vesicles and by the percentage of AM fungal root colonization. The AM fungal root traits were evaluated under an light microscope (Leitz, Labourlux S, Wetzlar, Germany) after root clearing and staining (Phillips and Hayman 1970) and using the modified grid-line intersect method (McGonigle et al. 1990). 2.5 Grain yield and nutrient uptake At physiological maturity (GS90), grain yield was determined by oven drying at 65 °C up to a constant weight. The concentration of N and P in the grains was determined by the Kjedahl method (Jones et al. 1991) and the ammonium-molybdophosphoric blue color method (Chapman and Pratt 1961), respectively. Furthermore, the grain concentration of K, Ca, Mg, Cu, Fe, Mn and Zn was determined by a microwave-assisted acid digestion system (COOLPEX Smart Microwave Reaction System, Yiyao Instrument Technology Development Co., Ltd., Shanghai, China) and a Microwave Plasma Atomic Emission Spectroscopy (4210 MP-AES, Agilent Technologies, Santa Clara, CA, USA). Host benefits were calculated (Avio et al. 2006). Moreover, in 2021, shoot samples at GS14 were oven dried at 65 °C up to a constant weight and the concentration of N and P was determined, according to the Kjedahl method (Jones et al. 1991) and the ammonium-molybdophosphoric blue color method (Chapman and Pratt 1961), respectively. 2.6 Mycorrhizal diversity in barley roots At both growth stages (GS14 and GS90), a root subsample per each replicate plot of the field experiment carried out in 2021 was prepared for DNA extraction, employing a combination of washing and ultrasound treatments to simultaneously separate the rhizospheric fraction (1 mm root soil attached) from roots and the roots colonized by endophytes (Bulgarelli et al. 2015). Genomic DNA was extracted from root samples (1 g of fresh weight) using the Dneasy Plant Mini Kit (Qiagen, Germany) (three barley genotypes x three replicate plot x two AMF inoculation level x two growth stages = a total of 36 samples). Details about the nested PCR approach applied are given in Supplementary Material and methods 2. The cleaned and quantified PCR products (a total of 18 per each growth stage) were adjusted in an equimolar ratio (10 ng μl -1 ) for the addition of dual-index barcodes using the Nextera® XT DNA library preparation kit (Illumina Inc., CA, United States). The generated metabarcoding libraries were sequenced on an Illumina MiSeq sequencer (2 × 300 bp paired-end reads) at the University of York (UK), loading a 12-pM final library concentration with 20% PhiX library spike-in (Illumina) and using an Illumina MiSeq V3 600 cycle sequencing kit. 2.7 Statistical analysis and bioinformatics A three-way analysis of variance (ANOVA) was performed to test the effect of year of cultivation (Y), wheat genotype (G) and AM fungal inoculation (Inoc) on the mycorrhizal abundance in barley roots, grain yield and nutrient concentration. Genotype and Inoc were considered as fixed factors and Y as a random factor. Data were transformed if necessary (e.g. log10, arcsen). The Tukey-B procedure was used to test the differences between means. The means and standard errors given in the tables and figures are for untransformed data. A multivariate approach based on permutational analysis of variance (PERMANOVA) was performed to test the effect of Y, G and Inoc and their interactions on plant and AM fungal parameters (Anderson 2005). Data in the matrix were square root transformed, standardized, and Euclidean distance matrices were calculated. Since PERMANOVA was statistically significant, principal coordinate analysis (PCO) was performed to visualize the most relevant patterns in the data (Gower 1966). In the PCO biplot, the overlay of vectors is reported. The analysis of homogeneity of multivariate dispersion (PERMDISP) (Anderson 2006) was performed to check the homogeneity of dispersion among groups (beta-diversity) (Anderson et al. 2006). To understand the relationship between the AM fungal abundance in roots and plant parameters at GS90 in the two years of cultivation, a multivariate statistical approach (RELATE analysis) was applied to determine the strength of the correlation between the two matrices in rank-order patterns of dissimilarity (Clarke and Warwick 2001). The analysis was based on the Spearman rank and 999 permutations with ρ equal to 1 representing the perfect relationship, and the result was plotted as a graph. Since the RELATE analysis was significant, BEST analysis was used to identify the main AM fungal traits responsible for plant functional changes. The BEST analysis was based on BioEnv methods (all combinations), Spearman rank, and 999 permutations (Clarke et al. 2008). Raw sequence data generated from the Illumina MiSeq sequencing run of the 36 samples (2021 year of cultivation: 18 samples per growth stage) were processed and analyzed using the QIIME2 (2018.11) pipeline and plugins (Bolyen et al. 2019). Demultiplexed forward and reverse paired-end reads were joined using the ‘-fastq_mergepairs’ of the USEARCH plugin (Edgar 2010). Details about bioinformatics are given in Supplementary Material and methods 3.. The resulting OTUs were assigned to virtual taxa (VTXs) using the MaarjAM database (https://maarjam.botany.ut.ee). All representative newly generated sequences were deposited in the NCBI Sequence Read (SRA) database as SUB14254691 (accession numbers from PP341529 to PP341554). Representative sequences were aligned with NCBI sequences of closely related AM fungal species (26 representative sequences and 17 NCBI sequences, for a total of 43 sequences), using the MAFFT online service (Katoh et al. 2019), and a Neighbor-Joining (NJ) tree was built using MEGA11 (Tamura et al. 2021), following the bootstrap test of phylogeny with 1,000 bootstraps. The substitution model used was the Kimura 2-parameter with uniform rates among sites, pairwise deletion, and 7 threads. The NJ tree was edited using Adobe Illustrator 2022. The AMF richness (S) was calculated as the number of VTXs per sample. Shannon index ( H ’) and Simpson index (λ) were also calculated (Supplementary Material and methods 4). To test the effect of G, Inoc, and growth stage (GS) on S, H ’ and λ, a three-way ANOVA was performed. Genotype, Inoc, and GS were considered as fixed factors. The data were transformed if necessary (i.e. log10). The differences between means were determined using the post-hoc Tukey-B procedure. The means and standard errors given in the tables are for untransformed data. All univariate analyzes were performed using the SPSS 25.0 software package (SPSS Inc., Chicago, IL, USA). To test the effect of G, Inoc, and GS and their interactions on the AM fungal community structure (relative abundances of AM fungal VTXs) a PERMANOVA analysis was carried out (Anderson 2005). Details are given in Supplementary Material and methods 5. The explained variance was calculated and divided among the sources of the variation. PERMDISP was applied (Anderson 2006). Data were plotted, according to the significance of PERMANOVA, using a non-metric multidimensional scaling (nMDS) (Kruskal 1964). In the nMDS plot, the AMF VTXs with a strong correlation ( r > 0.6) are displayed. The dataset was also used to generate Venn diagrams, representing the VTXs unique and shared to each treatment (i.e. AM fungal community composition; data in the Venn diagrams are expressed as percentages). The Venn diagrams were generated using InteractiVenn (Heberle et al. 2015) and edited by Adobe Illustrator 2022. To understand the relationship between the composition and the structure of the AM fungal community and plant parameters at GS90 and to identify the main AM fungal taxa responsible for plant functional changes, the RELATE analysis was applied as described above (Clarke and Warwick 2001). Since the RELATE analysis was significant between AM fungal structure at GS90 and plant parameters of the 2021 year of cultivation, plant parameters were also visualized, according to the significance of PERMANOVA (Anderson 2005), using a PCO (Gower 1966). The AM fungal community structure at GS90 was visualized by a nMDS (Kruskal 1964). In the nMDS plot, the AMF VTXs with a strong correlation ( r > 0.6) are displayed. The result of the RELATE analysis was plotted as a graph. Moreover, the best descriptor of the relationship was determined by the BEST analysis, as described above (Clarke et al. 2008). All multivariate analyzes were performed using PRIMER 7 and PERMANOVA + software (Anderson et al. 2008; Clarke and Gorley 2015). 3. Results and discussion 3.1 Abundance of AMF in roots of barley At physiological maturity (GS90), AM fungal root colonization and percentage of root length cointaining arbuscules were significantly affected by the interaction among year (Y), genotype (G) and AM fungal inoculation (Inoc) (Table S3). In 2020, Inoc increased the AM fungal root colonization of Atomo and Concerto up to an average of 53% (Fig.3a). Compared with not-inoculated plants (-M), the increases were 27% in Atomo and 10% in Concerto, whereas in Atlante there was a slightly, but not significant promotion. Moreover, in 2020, the variety Atomo showed the highest rate of arbuscules in roots (14%) and compared with the other treatments the increase was 12% (Fig. 3a,b). In 2021, a different pattern was reported, AM fungal root colonization and arbuscules values were generally higher than in 2020 (76% vs 41% and 64% vs 4%) and only Concerto showed significant increases of both AM fungal traits in comparison to control (+17 and +25, respetively) (Fig. 3a,b). Thus, the pattern of response of Concerto was consistent across the years of cultivation. The response of barley in term of AM fungal abundance was similar to values previously recorded in controlled sterile conditions with several spring cultivars and with many types of AM fungal inoculants (average AM fungal root colonization: 54%) (Baon et al. 1993; Coccina et al. 2019; Gray et al. 1991; Jensen 1983; Thirkell et al. 2019; Vierheilig et al. 2000; Watts-Williams et al. 2020). The responses in pot culture were recorded under a range of soil nutrient availability and water stress. However, similar to our results, also in controlled conditions, the cultivar affected AM fungal root colonization with a large range of response, from ca. 18% to 80%. Noteworthy, under water stress, the response in AM fungal colonization to inoculation was higher than in well-watered conditions (Jerbi et al. 2022). Positive AM fungal colonization responses following inoculation were consistenly observed under low soil P availability (Brown 2013; Jensen et al. 1984). Air temperature also played a significant role in AMF-barley interaction and low temperatures reduced the rate of AM fungal colonization in spring and winter cultivars compared to control temperatures (Hajiboland et al. 2019). These evidences can explain the different results we obtained in the two years. Indeed, in 2020, when soil was clay loam with very low P availability and no drought stress conditions, two over three varities of barley (i.e. Atomo and Concerto) were responsive to inoculation in term of AM fungal traits. By contrast, in 2021, with silty clay loam soil with low P availability and drough stress, only the variety Concerto was responsive to inoculation. Under field conditions, few experiments were carried out on barley and for more than one year, and no one tested different genotypes (Beslemes et al. 2023; Clarke and Mosse 1981; Heydari et al. 2023; Khaliq and Sanders 2000; Powell et al. 1980). Beslemes et al. (2023), in contrast with our results, did not find interactions between AM fungal inoculation and year of cultivation. Indeed, they found consistent increases between years of cultivation in AM fungal colonization in comparion with the control (+M vs -M: 55% vs 49%). Furthernore, our data highlighted an opposite pattern between years of cultivation in term of occurrence of vesicles in roots: in 2020 when AM fungal colonization and arbuscules were low, AMF invested their resources in vesicles, whereas in 2021 when AM fungal colonization and arbuscules were high, low percentages of vesicles were recored (Fig. 3c). 3.2 Effectiveness of AMF on barley grain yield Barley varieties responded differently to AM fungal inoculation in the two years of cultivation in terms of grain yield and nutrient concentration (Table S3). In 2020, according to the recorded positive response of AM fungal root colonization and arbuscules, grain yield was significantly promoted by 64% and 37% in the inoculated varieties Atomo and Concerto, respectively. Furthermore in line with no changes in AM fungal root traits, a slight but not significant enhancement was reported in Atlante (7%) (Fig. 4b). In 2021, Concerto showed a consistent positive response in grain yield (+78%) (Fig. 3a,b; Fig. 4a). This was also supported by the recorded promotion of root colonization and arbuscules. Moreover, while Atlante showed strong increases in grain yield under inoculation (+134%), Atomo did not show grain yield increases. Thus, the variety Concerto had a more stable response to inoculation with the indigenous AM fungal consortium and can be a good candidate for AM fungal inoculation, irrespective to soil nutrient availability and drought stress. Previously, in a meta-analysis on the effect of AM fungal inoculation on cereal grain yields, no effect was reported on barley both in controlled and field conditions (Zhang et al. 2019). However, they pointed out the lack of experiments on barley and the importance to study the effect of breeding and environment to validate the pattern. Under low soil nutrient availability, yield grain increases were reported in field inoculation with a mixture of AMF (Beslemes et al. 2023; Masrahi et al. 2023). In accordance with the general relationship found in crop plants between ∆AM and MR yield (Lekberg et al. 2005; Pellegrino et al. 2015), in 2020 we found a significant relationship (R 2 =0.71; P =0.004). By contrast, no relationship was found in 2021 (R 2 =0.024; P =0.691). This is in contrast with our expectations that higher AM fungal colonization would have greater driven grain yield under drought stress. However, our results support that soil nutrient availability are key drivers of the interaction AM fungal inoculation and crop yield (Zhang et al. 2019). 3.3 Effectiveness of AMF on barley nutrient uptake Inoculation determined in both years a consistent increase of P in grain of the three studied varieties (Fig. 4d; Table S3). However, the relative increase in 2021 (42%), under low soil P availability and drought stress, was larger than in 2020 (24%), characterized by a very low soil P availability and no drought stress. Thus, we can assert that under low and very low soil P availabilities, AM fungal inoculation in field conditions strongly promote the concentration of P in barley grains. Moreoverwe can assert that the symbiosis is more efficient under drought conditions. Nevertheless, the ∆AM and mycorrhizal P response ratio were not related between each others in both years (R 2 =194; P =0.236; R 2 =0.361; P =0.087). Furthermore, the positive and significant relationship we found between grain yield and P concentration (Fig. 4g) does not support a diluition effect mediated by AMF. By contrast to our results, a meta-analysis on the field inoculation did not find significant changes of P concentration in wheat grains (Pellegrino et al. 2015). However, the variability we observed between years is in accordance with Porcel and Ruiz-Lozano (2004) who reported that AMF enable host plants to grow and uptake P more efficiently under drought stress, through plant osmotic adjustment (Harrier 2001). As regard N concentration in grain, at both years of cultivation, Atomo and Atlante were reported to be positively affected by AM fungal inoculation (+6% and +8%, respectively), while Concerto was not affected in 2020 and negatively affected in 2021 (Fig. 5d). This result can not be explained by significant relationships between ∆AM and mycorrhizal N response ratio (data not shown). Moreover, grain yield and N concentration were not significant related (Fig. 4g). Thus, as previously observed in some genotypes of wheat, the consistent increases in Atomo and Atlante could be explained by a change of AM fungal communities in roots induced by inoculation (Marrassini et al. 2023). In several experiments, no changes in N grain concentration were reported in wheat (Pellegrino et al. 2015). However, the number of meta-analized field trials was low pointing out that field response of cereals to AMF deserves more study. Indeed, Glomus sp. and Gigaspora sp. and a multiple-species AM fungal field inoculum promoted the uptake of N in grain of two cultivars of barley (Beslemes et al. 2023; Masrahi et al. 2023). In 2021, the concentration of P was also promoted in the shoots of Concerto sampled at the four-leaves unfolded stage (GS14) (+18%) (Fig. S1a). This pattern of response to the AM fungal inoculation can support the success of the inoculation and the high responsiveness of this variety. Actually, this reponse is in line with the increase of AM fungal abundance in the roots of Concerto and with its grain yield response. In addition, averaged over genotypes, N shoot concentration was increased in inoculated treatments (Fig. S1b), further supporting the success of inoculation at early plant growth stages. The response in K uptake of barley genotypes to AM fungal inoculation was consistent among years (Fig. 5h). Atomo was the sole responsive variety (+12%). Overall, considering the two years, no relationship was observed between the ∆AM and mycorrhizal K response ratio (R 2 =0.000; P =0.939), and no relationship was underlighted between grain yield and K concentration (Fig. 4g). The increases of K observed in Atomo was similar to the results previously recorded on barley shoots and grain under field inoculation (Powell et al. 1980; Mahrahi et al. 2023). In addition, irrespective of inoculation, the three varieties showed higher grain K concentration in 2021 than 2020 (Fig. 5i). This is probably linked to differences in soil fertility. Moreover, we did not observe any effect on Mg concentration in grain due to AM fungal inoculation, with the exception of the promotion in 2020 with Atomo and Atlante (+39% and +17%, respectively). Grain Mg concentration was significantly and positively related to yield (Fig. 4g). Therefore, we can support an indirect effect of AMF. By contrast, not significant relationships were found between the ∆AM and mycorrhizal Mg response ratio (2020: R 2 =0.432, P =0.054; 2021: R 2 =0.091, P =0.430). Taking into consideration that K/Mg ratios is important for the nutritional status of plants(Xie et al. 2021), the fact that Mg and K were promoted in Atomo, could be an indicator of efficient physiological processes. Inoculation with AMF increased the concentration of Zn in grain of Atlante and Atomo, irrespective of the year of cultivation (+10% and +9%, respectively), while in Concerto no effect was observed (Fig. 5b). This response was not mediated by the increase of grain yield (Fig. 4g). Our results confirmed the general pattern of Zn response of crops, including wheat, corn and rice, to AMF (Lehmann et al. 2014; Pellegrino et al. 2015), altough no relationship was observed between the ∆AM and mycorrhizal Zn response ratio (R 2 =0.035; P =0.457). Nevertheless, our data did not confirm that the response is modulated by the availability of Zn in soil, since it did not vary between years of cultivations. A positive effect of R. irregularis was reported on shoot and grain Zn concentrations of barley when soil was fertilised with Zn (Al Mutairi et al. 2020; Cardini et al. 2021; Watts-Williams and Cavagnaro 2018). However, in accordance with our results, the AMF-mediated effect depended on barley genotypes (Al Mutairi et al. 2020). We also highlighted a significant interaction between barley genotype and year of cultivation (Table S3). Overall grain Zn concentration was higher in 2021 when soil had an adequate level of Zn availability than in 2020 when soil had very low Zn availability (Fig. 5c). However, the relative increase of Zn uptake into grain varied among barley varieties, and Concerto that in general uptakes less Zn in grain showed a relative lower increase (2021 vs 2020) respect to the other genotypes (Fig. 5b). Our data are in agreement with the results of a large study on the variability of grain Zn concentration in many wheat cultivars (Fan et al. 2008). The variety Concerto showed also a consistent increase of Fe in grain in 2020 when soil had an optimum level of Fe and in 2021 when soil had a low Fe availability (+24% and +130%, respetively) (Fig. 5a). Moreover, in 2020, Atomo showed, similarly to root colonization and yield responses, an increase in grain Fe concentration following inoculation (+140%). By contrast, in 2021, Atomo did not show changes in grain Fe concentration, similarly to the responses in root colonization and yield. Finally, no changes in grain Fe uptake were recorded in Atlante following inoculation, according to root colonization in both years (Fig. 5a). The positive mycorrhizal effect observed on barley Fe uptake is in line with the significant and positive relationship between the ∆AM and the Fe mycorrhizal response ratio found in 2020 and 2021 (R 2 =0.449, P =0.048; R 2 =0.590, P =0.016). Moreover, the increase of Fe concentration in grain were not determined by yield reduction (Fig. 4g). Overall, our results are in line with the positive effect reported by Lehmann and Rillig (2015) on crops (e.g., grasses) in lab and field conditions and with the large variability observed among wheat genotypes (Pellegrino et al. 2020). Arbuscular mycorrhizal fungal inoculation did not determine any changes on Mn uptake in barley grain or determined significant decreases (i.e., in 2020: -19% and -34% in Atomo and Concerto, respectively) (Fig. 5j). This is in accordance with the significant and negative relationship observed between the ∆AM and the mycorrhizal Mn response ratio (R 2 =0.464; P =0.043). This is in accordance with the overall decrease (-4%) under AM fungal inoculation (Lehmann and Rillig 2015). Explanations could be the fact that AMF increases soil pH and the availability of Mn in soil with subsequent leaching, and the reduction of Mn-reducing microbes or the promotion of Mn-oxidizing microbes. The concentration of Cu in grain was not affected by AMF, with the exception of Atlante that showed significant increases irrespective to year (+8) (Fig. 5k). This is in agreement with the not significant relationship observed between the ∆AM and the mycorrhizal Cu response ratio (R 2 =0.292; P =0.133). Moreover, our results are partially in agreement with the positive AM-fungal mediated effect found on crop Cu uptake (+29%) (Lehmann and Rillig 2015). However, since the Cu response to AMF was reported to be modulated by the availability of Cu in soil, with reductions at high availabilities, we can suppose that the availability of Cu in our soil was (very) low, and thus the response in Atomo and Concerto was undetectable. Moreover, since the year of cultivation variably affected the response of barley genotype in grain Cu uptake, irrespective of AM fungal inoculation, we can state that genotype in interaction with soil Cu availability could have determined these changes (Fig. 5l). Increases in Ca uptake in grain were observed, irrespective to year of cultivation, in Atlante and Concerto (+21% and 95%) (Fig. 5e). A direct effect of AMF could be hypothesized, since the uptake was not determined by yield increases (Fig. 4g). However, we can not directly link the AM abundance in roots with Ca uptake, since no significant relationship was observed between the ∆AM and the mycorrhizal Ca response ratio (R 2 =0.028; P =0.508). Calcium changes under AM fungal inoculation was scarcely investigated, and Watts-Williams and Gilbert (2021) found no changes in barley inoculated with R. irregularis . Furthermore, we did not observe any changes among barley varieties in 2021, whereas in 2020 Atomo and Concerto showed a higher uptake of Ca (Fig. 5f). In 2020, soil with a higher CEC and lower Ca availability could explain the interaction. Overall, our results support the fact that AMF can contribute to food nutrition. Finally, PERMANOVA allowed to summarize the pattern of plant and AM fungal parameters and to highlight significant interactions between Y, G and Inoc (Table S4). The PCO biplot (Fig. 6) showed that all genotypes differently responded to inoculation in the two year of cultivation. Although the year of cultivation was very strongly affecting the pattern of reponse, with an explained variance of 65%, the third-order interaction explained 7%. Looking at the PCO biplot, it showed a clear difference between years. In 2021 the agronomic reponse was less variable among replicate plots, and this encouraged a further investigation on root AM fungal communities. Our results are in contrast with our first hypothesis that genotype exerts a greater control over the response of barley to AM fungal inoculation than the environment. Indeed, our hypothesis was based on recent findings that wheat genotype inoculated in the field with the same indigenous consortium was a major driver of the agronomic response (Marrassini et al. 2024). This supports a less variable response of wheat. The PERMANOVA pairwise comparisons, utilised to dissect this interaction, highlighted consistent differences among the inoculated and not inoculated group of varieties and in 2020 and in 2021 (Table S5). Concerto was consistently and positively affected by AM fungal inoculation in both years, while Atomo and Atlante only in 2020 and 2021, respectively. This supports a robust and stable response of Concerto to inoculation across years and demostrates its less susceptibility to pedo-climatic variability. Therefore, the modern crossbreed variety Concerto developed in UK, showed a high mycorrhizal responsiveness to the indigenous AMF and this highlight the a good local adaptation of the crop to soil and AMF, regardless their origin. Furthermore, the year was consistently found to significantly affect the pattern of plant and AM fungal parameters in both inoculated and not-inoculated groups of barley varieties, underlining the major role of environment in shaping plant response under the same agronomic practice. Finally, PERMDISP showed significant difference among barley genotypes (Table S4). A higher variable pattern of response was observed in Atomo, whereas less variable patterns in Concerto and Atlante (Fig. 6). Therefore, irrespective to inoculation and year of cultivation, the response of Concerto and Atlante are more stable. 3.3. Diversity of AMF in the roots of barley To test our hypothesis that host plant preference in the AMF colonizing plant genotypes inoculated with indigenous AMF is driven by changes of community structure and not by changes in composition of AMF, we investigated the AM fungal community diversity in roots of the three barley genotypes. The study allowed also to investigate host plant preference under no inoculation. Moreover, we investigated the role of plant growth stage on modulating host plant preference. We focused the investigation on root samples collected in 2021. 3.3.1. Illumina sequencing output, AM fungal richness and alpha-diversity in barley roots After curation of the AM fungal sequences, 54,214 reads, ranging from 535 to 2,507 reads per sample, were retrieved and assigned to 26 VTXs (Fig. S2; Table S6). The 26 AMF VTXs belonged to three orders (i.e., Diversisporales, Entrophosporales, Glomerales) and four families (i.e., Diversisporacea, Gigasporaceae, Entrophosporaceae, Glomeraceae). The accumulation curves and rarefaction analyses of AMF confirmed that the Illumina sequencing effort was sufficient for the analysis (data not shown). Barley genotype and inoculation differently affected AM fungal richness (S) and alpha-diversity (H’ and λ) in the two growth stages (G12 and GS90) (Table S7). Atlante showed a higher diversity (S and H ’) than the other genotypes (+51% and +22%, respectively) (Table S8). Previously, the AM fungal diversity was only studied in single varieties of barley utilizing the taxonomic-based assessment of spores in soil and the molecular characterization in roots (Aguilera et al., 2017; Kaidzu et al. 2020). Therefore, our study highlights for the first time a host plant preference in AMF across barley genotypes. This is in accordance with the results obtained among genotypes of other crops (Kavadia et al. 2020; Mao et al. 2014; Parvin et al. 2021). Indeed, root traits variability among varieties of cereals, including barley, can be large (Nakhforrosh et al., 2014; Sendek et al. 2019; Robinson et al. 2018). Therefore, differences in root architecture could have determined the observed variable pattern of AM fungal diversity. However, Inoc increased AM fungal richness and H ’ only in Atlante at GS14 (+133% and +75%, respectively), while λ was promoted in Atlante and Atomo only at GS14 (+54% and +68%). In addition, AM fungal richness was higher at GS90 than GS14 (+23%). Since AM fungal diversity in roots promote plant productivity by the promotion of the abundance of productivity-promoting AMF (van der Heijden et al. 1998; Vogelsang et al. 2006), when inoculation determines the promotion of the diversity traits (i.e., Atlante and Atomo), a benefit of productivity can be expected. 3.3.2. Community composition and structure of AMF in barley roots Sixty-five percentage of AM fungal taxa (VTXs) (65%) were shared among genotypes, irrespective to AM fungal inoculation and plant growth stage (GS). Similarly, barley genotypes shared 52% and 50% VTXs in inoculated conditions and in control, irrespective to GS. Moreover, at GS14 the percentage of VTXs shared in inoculated conditions was similar to the one in the controls (32% and in 37%), while at GS90 it reached 65% in inoculated plants and 40% in controls. These results support our hypothesis of a good host preference in AMF across genotypes of barley in both inoculation treatments. They are in accordance with the variability in AM fungal composition observed in field across three wheat genotypes cultivated in a similar climatic area (Marassini et al., 2024; Pellegrino et al. 2020). Thus, also in barley there is a core composition of AMF that can be considered generalists, but there are several specialist taxa uniquely retrieved in the roots of each genotype. Moreover, since inoculation with indigenous AMF did not modify the rate of shared taxa at early stage, we can state that locally-sourced inocula did not change the AM fungal recruitment of the crop. By contrast, the increase of shared VTXs observed at GS90 due to inoculation, allows to highlight that indigenous AM fungal inoculants can reduce the variability in host preference among genotypes. Similarly, inoculating the same AM fungal consortium in wheat, the percentange of shared VTXs at maturity was higher than in the controls (46% vs 35%) (Marrassini et al. 2024). This supports the idea that indigenous taxa contained in the inoculum, during crop growth, colonize a larger number of plant genoypes and that our consortium was composed by high-compatible taxa. Focusing on the pattern of AM fungal composition in inoculated and not-inoculated barley genotypes, the average percentage of shared VTXs was 51% and 65% at GS14 and GS90, while the percentage of VTXs uniquelly retrieved in inoculated plants was 23% and 18%, respectively. Thus, we can not fully confirm our hypothesis that inoculation with a local AM fungal consortium did not modify the composition of the AM fungal communities. However, we can assume that when indigenous inoculants are applied they start to compete with closely related AM fungal species present in the soil at very low abundance, and thus not detectable by the molecular tools. Indeed, when individuals are rare in a microbial community, Illumina sequencing has some limitations in the accurancy of detection (Cheng et al. 2023; Egan et al. 2018). Moreover, the VTXs uniquelly found in the inoculated barley genotypes had a very low abundance (0.6%). Our findings are in accordance with previous results obtained on bread wheat genotypes and in sunflower, inoculated with the same AM fungal consortium in similar climatic conditions (Arcidiacono et al. 2024; Marrassini et al. 2024). Therefore, our data supports the low environmental and ecological impact of local AM fungal inoculants. Thus, biofertilization with indigenous AMF should be considered a suitable practice for the management of cropping systems. Looking at AM fungal community structures in the roots of barley genotypes in the PCO plot and in the shade plot (Fig. 7a,b), we can observe the significant interaction between G, Inoc and GS. This is supported by the results of PERMANOVA that highlighted a high percentage of explained variance of the third order interaction (24%) (Table S9). Overall, at GS14, no differences were recorded among not-inoculated barley genotypes, while significant differences were recorded between inoculated Atlante and Atomo/Concerto (Table S10). At GS90, no differences were recorded among not-inoculated barley genotypes and neither among inoculated genotypes. Therefore, at early and late plant development and under no inoculation, host preference in AMF is more discriminated by the pattern of composition than by the pattern of the community structure. The same occured at plant maturity in inoculated genotypes. By contrast, at early plant development, in inoculated conditions, host preference is discriminated by both composition and community structure. These findings highlight that to study host preference in managed field crops it is important to take into account these two aspects during the growing season. Moreover, inoculation determined at GS14 changes in AM fungal community structure only in Atlante, while at GS90 in both Atlante and Atomo. This results support the high variability in AM fungal community structure among wheat genotypes (Mao et al. 2014; Marrassini et al., 2024; Pellegrino et al. 2020) and gave new insights in the mechanisms by which diversity within AM fungal populations is enhance or maintain in roots. Indeed, a genotype like Concerto whose AM fungal community structure was similar in inoculated and not-inoculated treatments and AM fungal abundace and yield were promoted, can be considered a suitable variety in biofertilization programs. Previously, other factors, such as soil phosphate availability and host species were identified as main determinant of the root AM fungal structure (Cavagnaro et al., 2005; Ehinger et al., 2009; Eom et al. 2000; Helgason et al. 2002; Vanderkoornhuyse et al. 2003). Finally, the fact that two barley genotypes, Atomo and Atlante, in not-inoculated and inoculated conditions, respectively, showed significant differences in the AM fungal community structure between growth stages, is in accordance with a previous work reporting differences along the plant cycle in bread wheat, oat and barley, inoculated and not with a AMF (Aguilera et al. 2021). The importance of the phenological stage in shaping AM fungal community structure in roots of wheat was also reported in field conditions (Marrassini et al. 2024) and and perennial and annual grasses and non-grasses species (Lingfei et al. 2005). The role of barley phenological stage was underlined also by PERMDISP (Table S9). The higher variability at the early development of barley can be attributed to mechanisms of competition among AMF within a community (Chagnon et al., 2013; Jansa et al. 2008;). 3.4. Modelling barley productivity against AM fungal abundance and structure in roots Our results showed that there was a significant relationship between the traits of AM fungal abundance in roots and barley productivity (RELATE: ρ=0.587; P =0.001) (Fig. 8) and the BEST analysis allowed us to highlight arbuscules as the best predictor of barley productivity. Therefore, as we hypothesized, under low soil fertility AM fungal colonization traits, such as the percentage of arbuscules, are key and consistent determinants of the productivity of barley. Similarly, in field conditions, arbuscules were positively and strongly correlated with all functional traits of sunflower grown at low and high soil fertility (Arcidiacono et al. 2024). The association between increased AM fungal root colonization and increased barley productivity is consistent with the results of meta-analytic works (Lekberg and Koide 2005; McGonigle 1988; Pellegrino et al., 2015; Treseder, 2013). Moreover, the agronomic response of barley was driven also by changes in the structure of the AM fungal community, as highlighted in Fig. S3 and by the significance of RELATE analysis (ρ=0.514; P =0.005) (Fig. 8c), and not by changes of composition (ρ=0.084; P =0.167; data not shown). This relationship is clear comparing the pattern of AM fungal community structure in roots of barley genotypes at GS90 displayed by the nMDS plot (Fig. S3a) and the pattern of yield and nutrient uptake displayed by the PCO biplot (Fig S. 3b). Therefore, we confirmed our hypothesis that changes in AM fungal community structures and not in composition determine barley productivity. Moreover, the BEST analysis highlighted the best predictors of barley productivity (ρ=0.44, P =0.046) that was Glomus sp. VTX00342, considering one descriptor (r=0.333), or the taxa VTX00342 with Septoglomus sp. VTX00064, considering two descriptors (r=0.406) (Fig. 8d). These BEST predictors were also highlighted in the nMDS plot (Fig. S3a). Indeed, Glomus sp. VTX00342 and Septoglomus sp. VTX00064, putative members of the indigenous AM fungal inoculum, were more present in inoculated than not-inoculated plots (+46% and +56%, respectively). Previously, changes in AM fungal community structure and not in composition were identified as main driver of crop productivity under inoculation with indigenous AMF (Arcidiacono et al., 2024; Marrassini et al., 2024; Pellegrino et al. 2022). Indeed, the inoculated indigenous strains, highly compatible with all barley genotypes, as well as with the local environmental conditions, are preferentially recruited by the roots. Conclusions The general positive outcome in barley productivity supports the use of local AMF for building future inoculant generations and their large inclusion in sustainable agriculture. These results are particularly important under the current context where most minerals are significantly decreased in grain due to the dilution effects caused by intensification of agricultural management and high-yielding plant breeding. However, the underlined effect of environment in modulating the response of barley can help in the selection of genotypes with stable AM fungal response in specific climatic conditions. This is of key importance given the current challenges related to climate change. Moreover, our results better diclose the mechanisms at the basis of the host preference. Therefore, we suggest the propagation of AM fungal consortia adapted to specific climatic contexts to boost AM fungal inoculum production and to reduce the potential ecological risks of the exotic AM fungal inoculants. Declarations Acknowledgments The authors thank Domenico D’Alessio for hosting the field experiments on the farm Rinnovamento Agricolo, Santa Luce, Pisa (Italy). We also thank Dr. Giusto Giovannetti of CCS Aosta for large-scale culturing of the AMF inoculum. Funding This work was funded by the European Agricultural Fund for Rural Development 2007-2013 for Tuscany (Italy) measure 16.1 (FERTIBIO project; CUP Artea 743548; project leader Dr. Elisa Pellegrino) and measure 16.2 (FERTIBIO project; CUP Artea 828090; project leader Prof. Laura Ercoli. Valentina Marrassini was supported by a PhD scholarship in Agrobiosciences (XXXVI cycle) from the Scuola Superiore Sant’Anna (SSSA, Pisa, Italy). Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper. Ethics approval Not applicable. Consent to participate Not applicable. Consent for publication All the authors whose names appeared on the submission approved the version to be published and agreed to be accountable for all aspects of the work in ensuring that the questions related to the accuracy of integrity of any part of the work were appropriately investigated and resolved. Data availability Data will be made available on request. Furthermore, DNA sequences are available in the NCBI Sequence Read (SRA) database as SUB14254691. Code availability Not applicable. Author’s contributions Conceptualization, supervision, methodology, resources and funding acquisition, LE and EP; methodology, investigation, formal analysis, VM; data curation, result interpretation, visualization, writing original draft, review, and editing: VM, EP; review and editing: AVAP, LE. All authors gave their final approval for publication. References Aguilera P, Marín C, Oehl F, Godoy R, Borie F. 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version\u003c/p\u003e","description":"","filename":"Fig.8.png","url":"https://assets-eu.researchsquare.com/files/rs-4314201/v1/31815743036d93774840b57b.png"},{"id":60554599,"identity":"23bf4ae4-e81b-42a6-80f9-f7fa2280c14d","added_by":"auto","created_at":"2024-07-18 06:07:27","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10428117,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4314201/v1/0b4d8d0d-018f-4202-a903-1f07eccacd45.pdf"},{"id":60553836,"identity":"539ec7fc-4c8d-41a0-8d75-3fd0748f64cd","added_by":"auto","created_at":"2024-07-18 05:59:17","extension":"docx","order_by":12,"title":"","display":"","copyAsset":false,"role":"supplement","size":3291833,"visible":true,"origin":"","legend":"","description":"","filename":"SupplemetarymaterialsMarrassinietal.docx","url":"https://assets-eu.researchsquare.com/files/rs-4314201/v1/bb524afde52e223ccb4abb91.docx"}],"financialInterests":"","formattedTitle":"Positive response of barley to an indigenous arbuscular mycorrhizal fungal (AMF) inoculant is modulated by genotype and environment through changes in AMF root abundance and community structure","fulltext":[{"header":"1. Introduction ","content":"\u003cp\u003eBarley (\u003cem\u003eHordeum vulgare\u003c/em\u003e L.) production had increased steadily for over 30 years, but it should improve by 50% or even more by 2050 to ensure global food security (Fischer et al. 2014). However, the high intensity and frequency of extreme events due to climate change has severely affected crop production (Mall et al. 2017; Meza et al. 2020; Trenberth et al. 2014). Drought is reported to cause strong reductions of barley biomass and yield (Guo et al., 2009; Ozturk et al., 2002; Rollins et al 2013; Sallam et al., 2019; Samarah 2005). In addition, a decline of mineral concentration in grain under rising CO\u003csub\u003e2\u003c/sub\u003e was highlithed (Gojon et al. 2022). \u003c/p\u003e\n\u003cp\u003eMoreover, high use of mineral fertilisers in combination with low crop nutrient use efficiency results in a severe environmental issue and a high economic weight for farmers (Chien et al., 2009; Mclaughlin et al., 1996). These issues can be addressed either through the implementation of management practices (Mhlanga et al., 2021; Pittelkow et al., 2015; Sharma et al., 2021), or by the plant breeding (Lammerts van Bueren and Struik, 2017). In such a context, microbial biostimulants could represent an efficient tool to support barley productivity, especially under abiotic stresses, and to reduce the use of mineral fertilisers (Larimer et al., 2010; Philippot et al., 2013; Sch\u0026uuml;tz et al., 2018; Veresoglou and Menexes, 2010). \u003c/p\u003e\n\u003cp\u003eAmong beneficial microbes, arbuscular mycorrhizal fungi (AMF) (Glomeromycota; Tedersoo et al. 2018) has been reported to improve in field conditions wheat grain yield by 20% (Pellegrino et al 2015). This was further confirmed by Zhang et al. (2019) who estimated an increase of 16%. Mixed-species AM fungal inocula determined higher grain yield than single species. The highest AM fungal contribution to grain yield was detected at low soil N and P availabilities and in sandy soils. Wheat grain and straw P concentration were promoted through field AM fungal inoculation by 7% and 19%, respectively, while grain N content and Zn concentration by 31% and 7% (Pellegrino et al., 2015). \u003c/p\u003e\n\u003cp\u003eBarley is considered to be non responsive to AMF (Grace et al. 2008). Nevertheless, colonization under controlled and field conditions has been consistently reported (e.g., Clarke and Mosse, 1981; Jensen 1982; Masrahi et al. 2023; Powell et al. 1980). A neutral effect of AMF on yield was reported by Zhang et al. (2019), but the authours based this result only on three experiments and highlighted the need for improving data collection. In field conditions, inoculation with \u003cem\u003eGigaspora margarita\u003c/em\u003e promoted plant growth of barley by 92% (Powell et al. 1980). Under low P soil availability, ear fresh weight was doubled, irrespective of inoculated AM fungal species (Clarke and Mosse, 1981). Inoculants composed by three exotic AM fungal species increased grain yield and P uptake by 27% and 35%, respectively, while no effect was reported with native AM fungal mixtures (Powell et al., 1981). By contrast, no changes were reported on grain yield and P uptake of barley inoculated with \u003cem\u003eFunneliformis mosseae\u003c/em\u003e (Khaliq and Sanders 2000). Field inoculation with \u003cem\u003eF. mosseae\u003c/em\u003e in not fumigated plots resulted in significant depressions of grain and straw yield (-20%) (Khaliq and Sanders 1998). Therefore, under field conditions the outcome of the AM fungal inoculation was reported to be variable. This can be due the several factors, such as the abundance of infective propagules in soil, plant-fungal compatibility, composition of the AM fungal inocula, availability of soil nutrients and climatic parameters (Verbruggen et al., 2012). \u003c/p\u003e\n\u003cp\u003eIntensive agriculture practices, such as high P or N fertilizer rate, ploughing and continuous monoculture, have been shown to negatively impact the abundance, diversity and functionality of AMF in soil (Verbruggen et al. 2010a,b). Indeed, land use intensification modified the composition of AM fungal communities which resulted dominated by few taxa within Glomerales with low crop functionality (Oehl et al. 2010; Pellegrino et al., 2014). Therefore, it clear that in soils with low biological fertility is important to improve the abundance and diversity of AMF through inoculation in order to maximize their benefits on crops (Yang et al. 2014). \u003c/p\u003e\n\u003cp\u003eThe variability in plant benefits to AMF was explained by the identity of plant host and fungus (Klironomos 2003; Maherali and Klironomos 2007; Mensah et al. 2015; Munkvold et al. 2004). Large differences were observed among genotypes/cultivars of wheat (Hetrick et al. 1993, 1996). By contrast, barley was less investigated. Al Mutairi et al. (2020), testing the effect of inoculation of \u003cem\u003eR. irregularis\u003c/em\u003e on five barley cultivars, observed that genotype was a very strong driver for biomass, yield and yield components. Therefore, the study of barley intraspecific response to AMF it is of current great importance. Moreover, since the interaction between cereals and AMF was modulated by environmental factors (Grey 1991; Jerbi et al. 2020; Marrassini et al. 2024; Pellegrino et al. 2015), there is the need to carry out multiyear field studies. \u003c/p\u003e\n\u003cp\u003eCommonly, commercial AMF inocula are composed of generalist single or few exotic AM fungal species, having low genetic variability and not always offering efficiency and stability when applied (Salomon et al. 2022). Some commercial inoculants failed to form mycorrhizal associations, which may be caused by low adaptation to local edaphic conditions (Schreiner 2007). Moreover, since AMF were generally considered mutualistic, there has been little concern over potential negative consequences of their introduction. Nevertheless, the evidence that mycorrhizal function can range from mutualistic to parasitic (Johnson et al. 1997; Jones and Smith 2004; Klironomos 2003) led to take into account the potential agroecological concerns of exotic AM fungal introduction (Schwartz et al. 2006). Several studies were successfully carried out in the field with indigenous AMF, inoculated as single or mixture, on many field crops, and indigenous AMF were often reported to be more beneficial and less agroecologically harmful than exotic strains (Jansa et al. 2008; Oliveira et al. 2005; Pellegrino et al. 2011; Pellegrino and Bedini 2014). \u003c/p\u003e\n\u003cp\u003eTherefore, in this work, we inoculated a indigenous AM fungal consortium on three barley varieties for two years of cultivation to dissect the effect of the interaction AMF, genotype and environment on barley productivity, removing the effect of potential changes in AM fungal composition due to inoculation with exotic AMF (Fig. 1). In the present work, the benefit of AM fungal inoculation was evaluated by assessing grain yield and nutrient concentration (Fig. S1). Abundance and the community composition and structure of AMF in roots were evaluated using morphological and molecular tools. The indigenous inoculum was composed many fungal species, isolated from soil located in the same agricultural area where the experiment was carried out. We tested the following hypothesis: (i) barley genotype exerts a greater control over the response of the plant to AMF than the environment; (ii) under inoculation with indigenous AMF, host plant preference in AMF colonizing plant genotypes is driven by changes of community structure and not by changes in the composition; (iii) plant growth stage has a role in modulating the preference of barley genotypes; (iv) increases in AM fungal abundance and changes in community structure and not in composition determine barley productivity.\u003c/p\u003e"},{"header":"2. Material and Methods (2019 vs 1463 in altro MS)","content":"\u003cp\u003e\u003cstrong\u003e2.1 Fungal and plant material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe AM fungi used as inoculant were a consortium of taxa originating from a local field (Pellegrino and Bedini, 2014). The AM fungal inoculant was composed of 14 species belonging to five families: \u003cem\u003eAcaulospora cavernata\u003c/em\u003e, \u003cem\u003eAcaulospora spinosa\u003c/em\u003e, \u003cem\u003eAcaulospora\u003c/em\u003e spp., \u003cem\u003eDiversispora spurca\u003c/em\u003e, \u003cem\u003eFunneliformis coronatum\u003c/em\u003e, \u003cem\u003eEntrophospora etunicata\u003c/em\u003e (syn. \u003cem\u003eClaroideoglomus etunicatum\u003c/em\u003e), \u003cem\u003eFunneliformis geosporum\u003c/em\u003e, \u003cem\u003eFunneliformis mosseae\u003c/em\u003e, \u003cem\u003eGlomus\u003c/em\u003e spp., \u003cem\u003eRhizophagus clarus\u003c/em\u003e, \u003cem\u003eRhizophagus irregularis\u003c/em\u003e, \u003cem\u003eScutellospora aurigloba\u003c/em\u003e, \u003cem\u003eScutellospora calospora\u003c/em\u003e and \u003cem\u003eSeptoglomus viscosum\u003c/em\u003e. Three barley varieties were tested: Atlante (six-row, intermediate growth habit), Atomo (two-row, winter growth habit), and Concerto (two-row, spring growth habit) (Limagrain Italia SpA, Fidenza, Parma, Italy). For details about barley varieties see Table S1. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e2.2 Experimental field site\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe experiment was carried out in 2020 and 2021 at the \u0026lsquo;Societ\u0026agrave; Cooperativa Rinnovamento Agricolo\u0026rsquo;, Santa Luce, Pisa, Tuscany (43 \u0026deg; 26\u0026rsquo;24\u0026rsquo;\u0026rsquo;N-10 \u0026deg; 29\u0026rsquo;48\u0026rsquo;\u0026rsquo;E; 37 m above sea level) in two adjacent fields. The soil of 2020 and 2021 showed difference in texture (clay loam and silty clay loam, respectively), but similar low nutrient availability (Table S2 and Supplementary Material and methods 1). \u003c/p\u003e\n\u003cp\u003eThe climate of the site is cold and humid Mediterranean (Csa), according to the K\u0026ouml;ppen-Geiger climate classification (Kottek et al. 2006) with a five-year average annual precipitations of 1033 mm, a 10-year average of annual maximum and minimum daily air temperature of 20.4 \u0026deg;C and 10.8 \u0026deg;C, respectively. During barley cropping cycle in 2020 (January-July), mean maximum and minimum temperatures and total precipitation were 20.4 \u0026deg;C, 10.2 \u0026deg;C and 373 mm, while in 2021 (March-July) 22.7 \u0026deg;C, 11.7 \u0026deg;C and 170 mm, respectively (Fig. 2).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e2.3 Experimental set-up and sampling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA complete factorial experimental design with three factors was adopted with two years of cultivation (2020 and 2021), three barley varieties, and two AM fungal inoculation treatments (inoculated with the AM fungal consortium, +M; not-inoculated/control, -M) (Fig. 1). The experiment was arranged in a completely randomized design with three replicate plots (8 m x 42 m = 336 m\u003csup\u003e2 \u003c/sup\u003ein 2020; 8 m x 20 m = 160 m\u003csup\u003e2 \u003c/sup\u003ein 2021). The inoculum, produced as described by Pellegrino and Bedini (2014), was a micronized mixture of mycorrhized roots of sorghum (\u003cem\u003eSorghum halepense\u003c/em\u003e L.), spores, hyphal fragments, and bentonite as carrier. The inoculum was distributed at the sowing by manual application to seeds that had been previously moistened with water. The rate of the AM fungal inoculum was 0.8 g m\u003csup\u003e2 \u003c/sup\u003e(8 kg ha\u003csup\u003e-1\u003c/sup\u003e) (1.2 kg of inoculum 100 kg\u003csup\u003e-1 \u003c/sup\u003eseeds, about 2500 spores ha\u003csup\u003e-1\u003c/sup\u003e). The mock inoculum (not-inoculated/control) consisted of the same dose of steam-sterilized AM fungal inoculum (121 \u0026deg;C for 25 min on two consecutive days). To ensure a common microflora, both inocula received 0.05 L kg\u003csup\u003e-1\u003c/sup\u003e of a filtrate obtained by filtering through a Whatman no. 1 filter paper the AM fungal consortium. The seed rate was 200 kg ha\u003csup\u003e-1\u003c/sup\u003e, corresponding to approximately 350 viable seeds per m\u003csup\u003e2\u003c/sup\u003e, distributed in rows 14 cm apart. Barley was seeded with a pneumatic seeding machine (Aguirre Bota) on 17 January 2020 and 1 March 2021. Before the experimental setup, the preceding crop was clover (\u003cem\u003eTrifolium\u003c/em\u003e \u003cem\u003ealexandrinum\u003c/em\u003e L.) in 2020 and faba bean (\u003cem\u003eVicia faba\u003c/em\u003e L. var. minor) in 2021. Soil tillage was carried out in autumn by moldboard plowing at a soil depth of 30 cm, and by harrowing at a soil depth of 15 cm, immediately before seeding. No organic/chemical fertilizer was applied. No weed and pest/pathogen control treatments were applied. In both years of cultivation at the stage of four leaves unfolded (GS14) (Zadoks et al. 1974) and at the physiological maturity (GS90), ten plants, randomly selected in each replicate plot, were excavated with their root system to determine AM fungal abundance and diversity. Barley was harvested on 22 July 2020 and 26 July 2021 in each replicate plot by a combine harvester (Laverda, Vincenza, Italy). Furthermore, in 2021, at GS14, shoots from the ten plants were also sampled. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e2.4 \u003c/strong\u003e\u003cstrong\u003eMycorrhizal abundance in barley roots\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt each sampling, fresh roots from each replicate plot were combined and cleaned from the attached soil by soft washing with tap water. Mycorrhizal abundance was measured by the percentage of root length containing arbuscules and vesicles and by the percentage of AM fungal root colonization. The AM fungal root traits were evaluated under an light microscope (Leitz, Labourlux S, Wetzlar, Germany) after root clearing and staining (Phillips and Hayman 1970) and using the modified grid-line intersect method (McGonigle et al. 1990). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e2.5 Grain yield and nutrient uptake\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt physiological maturity (GS90), grain yield was determined by oven drying at 65 \u0026deg;C up to a constant weight. The concentration of N and P in the grains was determined by the Kjedahl method (Jones et al. 1991) and the ammonium-molybdophosphoric blue color method (Chapman and Pratt 1961), respectively. Furthermore, the grain concentration of K, Ca, Mg, Cu, Fe, Mn and Zn was determined by a microwave-assisted acid digestion system (COOLPEX Smart Microwave Reaction System, Yiyao Instrument Technology Development Co., Ltd., Shanghai, China) and a Microwave Plasma Atomic Emission Spectroscopy (4210 MP-AES, Agilent Technologies, Santa Clara, CA, USA). Host benefits were calculated (Avio et al. 2006). Moreover, in 2021, shoot samples at GS14 were oven dried at 65 \u0026deg;C up to a constant weight and the concentration of N and P was determined, according to the Kjedahl method (Jones et al. 1991) and the ammonium-molybdophosphoric blue color method (Chapman and Pratt 1961), respectively.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e2.6 Mycorrhizal diversity in barley roots\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt both growth stages (GS14 and GS90), a root subsample per each replicate plot of the field experiment carried out in 2021 was prepared for DNA extraction, employing a combination of washing and ultrasound treatments to simultaneously separate the rhizospheric fraction (1 mm root soil attached) from roots and the roots colonized by endophytes (Bulgarelli et al. 2015). Genomic DNA was extracted from root samples (1 g of fresh weight) using the Dneasy Plant Mini Kit (Qiagen, Germany) (three barley genotypes x three replicate plot x two AMF inoculation level x two growth stages = a total of 36 samples). Details about the nested PCR approach applied are given in Supplementary Material and methods 2. The cleaned and quantified PCR products (a total of 18 per each growth stage) were adjusted in an equimolar ratio (10 ng \u0026mu;l\u003csup\u003e-1\u003c/sup\u003e) for the addition of dual-index barcodes using the Nextera\u0026reg; XT DNA library preparation kit (Illumina Inc., CA, United States). The generated metabarcoding libraries were sequenced on an Illumina MiSeq sequencer (2 \u0026times; 300 bp paired-end reads) at the University of York (UK), loading a 12-pM final library concentration with 20% PhiX library spike-in (Illumina) and using an Illumina MiSeq V3 600 cycle sequencing kit.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e2.7 Statistical analysis and bioinformatics\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA three-way analysis of variance (ANOVA) was performed to test the effect of year of cultivation (Y), wheat genotype (G) and AM fungal inoculation (Inoc) on the mycorrhizal abundance in barley roots, grain yield and nutrient concentration. Genotype and Inoc were considered as fixed factors and Y as a random factor. Data were transformed if necessary (e.g. log10, arcsen). The Tukey-B procedure was used to test the differences between means. The means and standard errors given in the tables and figures are for untransformed data. \u003c/p\u003e\n\u003cp\u003eA multivariate approach based on permutational analysis of variance (PERMANOVA) was performed to test the effect of Y, G and Inoc and their interactions on plant and AM fungal parameters (Anderson 2005). Data in the matrix were square root transformed, standardized, and Euclidean distance matrices were calculated. Since PERMANOVA was statistically significant, principal coordinate analysis (PCO) was performed to visualize the most relevant patterns in the data (Gower 1966). In the PCO biplot, the overlay of vectors is reported. The analysis of homogeneity of multivariate dispersion (PERMDISP) (Anderson 2006) was performed to check the homogeneity of dispersion among groups (beta-diversity) (Anderson et al. 2006). To understand the relationship between the AM fungal abundance in roots and plant parameters at GS90 in the two years of cultivation, a multivariate statistical approach (RELATE analysis) was applied to determine the strength of the correlation between the two matrices in rank-order patterns of dissimilarity (Clarke and Warwick 2001). The analysis was based on the Spearman rank and 999 permutations with \u0026rho; equal to 1 representing the perfect relationship, and the result was plotted as a graph. Since the RELATE analysis was significant, BEST analysis was used to identify the main AM fungal traits responsible for plant functional changes. The BEST analysis was based on BioEnv methods (all combinations), Spearman rank, and 999 permutations (Clarke et al. 2008). \u003c/p\u003e\n\u003cp\u003eRaw sequence data generated from the Illumina MiSeq sequencing run of the 36 samples (2021 year of cultivation: 18 samples per growth stage) were processed and analyzed using the QIIME2 (2018.11) pipeline and plugins (Bolyen et al. 2019). Demultiplexed forward and reverse paired-end reads were joined using the \u0026lsquo;-fastq_mergepairs\u0026rsquo; of the USEARCH plugin (Edgar 2010). Details about bioinformatics are given in Supplementary Material and methods 3.. The resulting OTUs were assigned to virtual taxa (VTXs) using the MaarjAM database (https://maarjam.botany.ut.ee). All representative newly generated sequences were deposited in the NCBI Sequence Read (SRA) database as SUB14254691 (accession numbers from PP341529 to PP341554). Representative sequences were aligned with NCBI sequences of closely related AM fungal species (26 representative sequences and 17 NCBI sequences, for a total of 43 sequences), using the MAFFT online service (Katoh et al. 2019), and a Neighbor-Joining (NJ) tree was built using MEGA11 (Tamura et al. 2021), following the bootstrap test of phylogeny with 1,000 bootstraps. The substitution model used was the Kimura 2-parameter with uniform rates among sites, pairwise deletion, and 7 threads. The NJ tree was edited using Adobe Illustrator 2022.\u003c/p\u003e\n\u003cp\u003eThe AMF richness (S) was calculated as the number of VTXs per sample. Shannon index (\u003cem\u003eH\u003c/em\u003e\u0026rsquo;) and Simpson index (\u0026lambda;) were also calculated (Supplementary Material and methods 4). To test the effect of G, Inoc, and growth stage (GS) on S, \u003cem\u003eH\u003c/em\u003e\u0026rsquo; and \u0026lambda;, a three-way ANOVA was performed. Genotype, Inoc, and GS were considered as fixed factors. The data were transformed if necessary (i.e. log10). The differences between means were determined using the post-hoc Tukey-B procedure. The means and standard errors given in the tables are for untransformed data. All univariate analyzes were performed using the SPSS 25.0 software package (SPSS Inc., Chicago, IL, USA).\u003c/p\u003e\n\u003cp\u003eTo test the effect of G, Inoc, and GS and their interactions on the AM fungal community structure (relative abundances of AM fungal VTXs) a PERMANOVA analysis was carried out (Anderson 2005). Details are given in Supplementary Material and methods 5. The explained variance was calculated and divided among the sources of the variation. PERMDISP was applied (Anderson 2006). Data were plotted, according to the significance of PERMANOVA, using a non-metric multidimensional scaling (nMDS) (Kruskal 1964). In the nMDS plot, the AMF VTXs with a strong correlation (\u003cem\u003er \u003c/em\u003e\u0026gt; 0.6) are displayed. The dataset was also used to generate Venn diagrams, representing the VTXs unique and shared to each treatment (i.e. AM fungal community composition; data in the Venn diagrams are expressed as percentages). The Venn diagrams were generated using InteractiVenn (Heberle et al. 2015) and edited by Adobe Illustrator 2022. \u003c/p\u003e\n\u003cp\u003eTo understand the relationship between the composition and the structure of the AM fungal community and plant parameters at GS90 and to identify the main AM fungal taxa responsible for plant functional changes, the RELATE analysis was applied as described above (Clarke and Warwick 2001). Since the RELATE analysis was significant between AM fungal structure at GS90 and plant parameters of the 2021 year of cultivation, plant parameters were also visualized, according to the significance of PERMANOVA (Anderson 2005), using a PCO (Gower 1966). The AM fungal community structure at GS90 was visualized by a nMDS (Kruskal 1964). In the nMDS plot, the AMF VTXs with a strong correlation (\u003cem\u003er \u003c/em\u003e\u0026gt; 0.6) are displayed. The result of the RELATE analysis was plotted as a graph. Moreover, the best descriptor of the relationship was determined by the BEST analysis, as described above (Clarke et al. 2008). All multivariate analyzes were performed using PRIMER 7 and PERMANOVA + software (Anderson et al. 2008; Clarke and Gorley 2015).\u003c/p\u003e"},{"header":"3. Results and discussion","content":"\u003cp\u003e\u003cstrong\u003e3.1 Abundance of AMF in roots of barley\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAt physiological maturity (GS90), AM fungal root colonization and percentage of root length cointaining arbuscules were significantly affected by the interaction among year (Y), genotype (G) and AM fungal inoculation (Inoc) (Table S3). In 2020, Inoc increased the AM fungal root colonization of Atomo and Concerto up to an average of 53% (Fig.3a). Compared with not-inoculated plants (-M), the increases were 27% in Atomo and 10% in Concerto, whereas in Atlante there was a slightly, but not significant promotion. Moreover, in 2020, the variety Atomo showed the highest rate of arbuscules in roots (14%) and compared with the other treatments the increase was 12% (Fig. 3a,b). In 2021, a different pattern was reported, AM fungal root colonization and arbuscules values were generally higher than in 2020 (76% \u003cem\u003evs\u003c/em\u003e 41% and 64% \u003cem\u003evs\u003c/em\u003e 4%) and only Concerto showed significant increases of both AM fungal traits in comparison to control (+17 and +25, respetively) (Fig. 3a,b). Thus, the pattern of response of Concerto was consistent across the years of cultivation. \u003c/p\u003e\n\u003cp\u003eThe response of barley in term of AM fungal abundance was similar to values previously recorded in controlled sterile conditions with several spring cultivars and with many types of AM fungal inoculants (average AM fungal root colonization: 54%) (Baon et al. 1993; Coccina et al. 2019; Gray et al. 1991; Jensen 1983; Thirkell et al. 2019; Vierheilig et al. 2000; Watts-Williams et al. 2020). The responses in pot culture were recorded under a range of soil nutrient availability and water stress. However, similar to our results, also in controlled conditions, the cultivar affected AM fungal root colonization with a large range of response, from ca. 18% to 80%. Noteworthy, under water stress, the response in AM fungal colonization to inoculation was higher than in well-watered conditions (Jerbi et al. 2022). Positive AM fungal colonization responses following inoculation were consistenly observed under low soil P availability (Brown 2013; Jensen et al. 1984). \u003c/p\u003e\n\u003cp\u003eAir temperature also played a significant role in AMF-barley interaction and low temperatures reduced the rate of AM fungal colonization in spring and winter cultivars compared to control temperatures (Hajiboland et al. 2019). These evidences can explain the different results we obtained in the two years. Indeed, in 2020, when soil was clay loam with very low P availability and no drought stress conditions, two over three varities of barley (i.e. Atomo and Concerto) were responsive to inoculation in term of AM fungal traits. By contrast, in 2021, with silty clay loam soil with low P availability and drough stress, only the variety Concerto was responsive to inoculation. Under field conditions, few experiments were carried out on barley and for more than one year, and no one tested different genotypes (Beslemes et al. 2023; Clarke and Mosse 1981; Heydari et al. 2023; Khaliq and Sanders 2000; Powell et al. 1980). Beslemes et al. (2023), in contrast with our results, did not find interactions between AM fungal inoculation and year of cultivation. Indeed, they found consistent increases between years of cultivation in AM fungal colonization in comparion with the control (+M \u003cem\u003evs\u003c/em\u003e -M: 55% \u003cem\u003evs\u003c/em\u003e 49%). \u003c/p\u003e\n\u003cp\u003eFurthernore, our data highlighted an opposite pattern between years of cultivation in term of occurrence of vesicles in roots: in 2020 when AM fungal colonization and arbuscules were low, AMF invested their resources in vesicles, whereas in 2021 when AM fungal colonization and arbuscules were high, low percentages of vesicles were recored (Fig. 3c).\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e3.2\u003c/strong\u003e \u003cstrong\u003eEffectiveness of AMF on barley grain yield \u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eBarley varieties responded differently to AM fungal inoculation in the two years of cultivation in terms of grain yield and nutrient concentration (Table S3). In 2020, according to the recorded positive response of AM fungal root colonization and arbuscules, grain yield was significantly promoted by 64% and 37% in the inoculated varieties Atomo and Concerto, respectively. Furthermore in line with no changes in AM fungal root traits, a slight but not significant enhancement was reported in Atlante (7%) (Fig. 4b). In 2021, Concerto showed a consistent positive response in grain yield (+78%) (Fig. 3a,b; Fig. 4a). This was also supported by the recorded promotion of root colonization and arbuscules. Moreover, while Atlante showed strong increases in grain yield under inoculation (+134%), Atomo did not show grain yield increases. Thus, the variety Concerto had a more stable response to inoculation with the indigenous AM fungal consortium and can be a good candidate for AM fungal inoculation, irrespective to soil nutrient availability and drought stress. \u003c/p\u003e\n\u003cp\u003ePreviously, in a meta-analysis on the effect of AM fungal inoculation on cereal grain yields, no effect was reported on barley both in controlled and field conditions (Zhang et al. 2019). However, they pointed out the lack of experiments on barley and the importance to study the effect of breeding and environment to validate the pattern. Under low soil nutrient availability, yield grain increases were reported in field inoculation with a mixture of AMF (Beslemes et al. 2023; Masrahi et al. 2023). In accordance with the general relationship found in crop plants between ∆AM and MR\u003csub\u003eyield\u003c/sub\u003e (Lekberg et al. 2005; Pellegrino et al. 2015), in 2020 we found a significant relationship (R\u003csup\u003e2\u003c/sup\u003e=0.71; \u003cem\u003eP\u003c/em\u003e=0.004). By contrast, no relationship was found in 2021 (R\u003csup\u003e2\u003c/sup\u003e=0.024; \u003cem\u003eP\u003c/em\u003e=0.691). This is in contrast with our expectations that higher AM fungal colonization would have greater driven grain yield under drought stress. However, our results support that soil nutrient availability are key drivers of the interaction AM fungal inoculation and crop yield (Zhang et al. 2019). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e3.3 \u003c/strong\u003e\u003cstrong\u003eEffectiveness of AMF on barley nutrient uptake\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eInoculation determined in both years a consistent increase of P in grain of the three studied varieties (Fig. 4d; Table S3). However, the relative increase in 2021 (42%), under low soil P availability and drought stress, was larger than in 2020 (24%), characterized by a very low soil P availability and no drought stress. Thus, we can assert that under low and very low soil P availabilities, AM fungal inoculation in field conditions strongly promote the concentration of P in barley grains. Moreoverwe can assert that the symbiosis is more efficient under drought conditions. Nevertheless, the ∆AM and mycorrhizal P response ratio were not related between each others in both years (R\u003csup\u003e2\u003c/sup\u003e=194; \u003cem\u003eP\u003c/em\u003e=0.236; R\u003csup\u003e2\u003c/sup\u003e=0.361; \u003cem\u003eP\u003c/em\u003e=0.087). Furthermore, the positive and significant relationship we found between grain yield and P concentration (Fig. 4g) does not support a diluition effect mediated by AMF. \u003c/p\u003e\n\u003cp\u003eBy contrast to our results, a meta-analysis on the field inoculation did not find significant changes of P concentration in wheat grains (Pellegrino et al. 2015). However, the variability we observed between years is in accordance with Porcel and Ruiz-Lozano (2004) who reported that AMF enable host plants to grow and uptake P more efficiently under drought stress, through plant osmotic adjustment (Harrier 2001). \u003c/p\u003e\n\u003cp\u003eAs regard N concentration in grain, at both years of cultivation, Atomo and Atlante were reported to be positively affected by AM fungal inoculation (+6% and +8%, respectively), while Concerto was not affected in 2020 and negatively affected in 2021 (Fig. 5d). This result can not be explained by significant relationships between ∆AM and mycorrhizal N response ratio (data not shown). Moreover, grain yield and N concentration were not significant related (Fig. 4g). Thus, as previously observed in some genotypes of wheat, the consistent increases in Atomo and Atlante could be explained by a change of AM fungal communities in roots induced by inoculation (Marrassini et al. 2023). In several experiments, no changes in N grain concentration were reported in wheat (Pellegrino et al. 2015). However, the number of meta-analized field trials was low pointing out that field response of cereals to AMF deserves more study. Indeed, \u003cem\u003eGlomus\u003c/em\u003e sp. and \u003cem\u003eGigaspora\u003c/em\u003e sp. and a multiple-species AM fungal field inoculum promoted the uptake of N in grain of two cultivars of barley (Beslemes et al. 2023; Masrahi et al. 2023). \u003c/p\u003e\n\u003cp\u003eIn 2021, the concentration of P was also promoted in the shoots of Concerto sampled at the four-leaves unfolded stage (GS14) (+18%) (Fig. S1a). This pattern of response to the AM fungal inoculation can support the success of the inoculation and the high responsiveness of this variety. Actually, this reponse is in line with the increase of AM fungal abundance in the roots of Concerto and with its grain yield response. In addition, averaged over genotypes, N shoot concentration was increased in inoculated treatments (Fig. S1b), further supporting the success of inoculation at early plant growth stages.\u003c/p\u003e\n\u003cp\u003eThe response in K uptake of barley genotypes to AM fungal inoculation was consistent among years (Fig. 5h). Atomo was the sole responsive variety (+12%). Overall, considering the two years, no relationship was observed between the ∆AM and mycorrhizal K response ratio (R\u003csup\u003e2\u003c/sup\u003e=0.000; \u003cem\u003eP\u003c/em\u003e=0.939), and no relationship was underlighted between grain yield and K concentration (Fig. 4g). The increases of K observed in Atomo was similar to the results previously recorded on barley shoots and grain under field inoculation (Powell et al. 1980; Mahrahi et al. 2023). In addition, irrespective of inoculation, the three varieties showed higher grain K concentration in 2021 than 2020 (Fig. 5i). This is probably linked to differences in soil fertility.\u003c/p\u003e\n\u003cp\u003eMoreover, we did not observe any effect on Mg concentration in grain due to AM fungal inoculation, with the exception of the promotion in 2020 with Atomo and Atlante (+39% and +17%, respectively). Grain Mg concentration was significantly and positively related to yield (Fig. 4g). Therefore, we can support an indirect effect of AMF. By contrast, not significant relationships were found between the ∆AM and mycorrhizal Mg response ratio (2020: R\u003csup\u003e2\u003c/sup\u003e=0.432, \u003cem\u003eP\u003c/em\u003e=0.054; 2021: R\u003csup\u003e2\u003c/sup\u003e=0.091, \u003cem\u003eP\u003c/em\u003e=0.430). Taking into consideration that K/Mg ratios is important for the nutritional status of plants(Xie et al. 2021), the fact that Mg and K were promoted in Atomo, could be an indicator of efficient physiological processes.\u003c/p\u003e\n\u003cp\u003eInoculation with AMF increased the concentration of Zn in grain of Atlante and Atomo, irrespective of the year of cultivation (+10% and +9%, respectively), while in Concerto no effect was observed (Fig. 5b). This response was not mediated by the increase of grain yield (Fig. 4g). Our results confirmed the general pattern of Zn response of crops, including wheat, corn and rice, to AMF (Lehmann et al. 2014; Pellegrino et al. 2015), altough no relationship was observed between the ∆AM and mycorrhizal Zn response ratio (R\u003csup\u003e2\u003c/sup\u003e=0.035; \u003cem\u003eP\u003c/em\u003e=0.457). Nevertheless, our data did not confirm that the response is modulated by the availability of Zn in soil, since it did not vary between years of cultivations. \u003c/p\u003e\n\u003cp\u003eA positive effect of \u003cem\u003eR. irregularis\u003c/em\u003e was reported on shoot and grain Zn concentrations of barley when soil was fertilised with Zn (Al Mutairi et al. 2020; Cardini et al. 2021; Watts-Williams and Cavagnaro 2018). However, in accordance with our results, the AMF-mediated effect depended on barley genotypes (Al Mutairi et al. 2020). We also highlighted a significant interaction between barley genotype and year of cultivation (Table S3). Overall grain Zn concentration was higher in 2021 when soil had an adequate level of Zn availability than in 2020 when soil had very low Zn availability (Fig. 5c). However, the relative increase of Zn uptake into grain varied among barley varieties, and Concerto that in general uptakes less Zn in grain showed a relative lower increase (2021 \u003cem\u003evs\u003c/em\u003e 2020) respect to the other genotypes (Fig. 5b). Our data are in agreement with the results of a large study on the variability of grain Zn concentration in many wheat cultivars (Fan et al. 2008). \u003c/p\u003e\n\u003cp\u003eThe variety Concerto showed also a consistent increase of Fe in grain in 2020 when soil had an optimum level of Fe and in 2021 when soil had a low Fe availability (+24% and +130%, respetively) (Fig. 5a). Moreover, in 2020, Atomo showed, similarly to root colonization and yield responses, an increase in grain Fe concentration following inoculation (+140%). By contrast, in 2021, Atomo did not show changes in grain Fe concentration, similarly to the responses in root colonization and yield. Finally, no changes in grain Fe uptake were recorded in Atlante following inoculation, according to root colonization in both years (Fig. 5a). The positive mycorrhizal effect observed on barley Fe uptake is in line with the significant and positive relationship between the ∆AM and the Fe mycorrhizal response ratio found in 2020 and 2021 (R\u003csup\u003e2\u003c/sup\u003e=0.449, \u003cem\u003eP\u003c/em\u003e=0.048; R\u003csup\u003e2\u003c/sup\u003e=0.590, \u003cem\u003eP\u003c/em\u003e=0.016). Moreover, the increase of Fe concentration in grain were not determined by yield reduction (Fig. 4g). Overall, our results are in line with the positive effect reported by Lehmann and Rillig (2015) on crops (e.g., grasses) in lab and field conditions and with the large variability observed among wheat genotypes (Pellegrino et al. 2020). \u003c/p\u003e\n\u003cp\u003eArbuscular mycorrhizal fungal inoculation did not determine any changes on Mn uptake in barley grain or determined significant decreases (i.e., in 2020: -19% and -34% in Atomo and Concerto, respectively) (Fig. 5j). This is in accordance with the significant and negative relationship observed between the ∆AM and the mycorrhizal Mn response ratio (R\u003csup\u003e2\u003c/sup\u003e=0.464; \u003cem\u003eP\u003c/em\u003e=0.043). This is in accordance with the overall decrease (-4%) under AM fungal inoculation (Lehmann and Rillig 2015). Explanations could be the fact that AMF increases soil pH and the availability of Mn in soil with subsequent leaching, and the reduction of Mn-reducing microbes or the promotion of Mn-oxidizing microbes. \u003c/p\u003e\n\u003cp\u003eThe concentration of Cu in grain was not affected by AMF, with the exception of Atlante that showed significant increases irrespective to year (+8) (Fig. 5k). This is in agreement with the not significant relationship observed between the ∆AM and the mycorrhizal Cu response ratio (R\u003csup\u003e2\u003c/sup\u003e=0.292; \u003cem\u003eP\u003c/em\u003e=0.133). Moreover, our results are partially in agreement with the positive AM-fungal mediated effect found on crop Cu uptake (+29%) (Lehmann and Rillig 2015). However, since the Cu response to AMF was reported to be modulated by the availability of Cu in soil, with reductions at high availabilities, we can suppose that the availability of Cu in our soil was (very) low, and thus the response in Atomo and Concerto was undetectable. Moreover, since the year of cultivation variably affected the response of barley genotype in grain Cu uptake, irrespective of AM fungal inoculation, we can state that genotype in interaction with soil Cu availability could have determined these changes (Fig. 5l).\u003c/p\u003e\n\u003cp\u003eIncreases in Ca uptake in grain were observed, irrespective to year of cultivation, in Atlante and Concerto (+21% and 95%) (Fig. 5e). A direct effect of AMF could be hypothesized, since the uptake was not determined by yield increases (Fig. 4g). However, we can not directly link the AM abundance in roots with Ca uptake, since no significant relationship was observed between the ∆AM and the mycorrhizal Ca response ratio (R\u003csup\u003e2\u003c/sup\u003e=0.028; \u003cem\u003eP\u003c/em\u003e=0.508). Calcium changes under AM fungal inoculation was scarcely investigated, and Watts-Williams and Gilbert (2021) found no changes in barley inoculated with \u003cem\u003eR. irregularis\u003c/em\u003e. Furthermore, we did not observe any changes among barley varieties in 2021, whereas in 2020 Atomo and Concerto showed a higher uptake of Ca (Fig. 5f). In 2020, soil with a higher CEC and lower Ca availability could explain the interaction. Overall, our results support the fact that AMF can contribute to food nutrition. \u003c/p\u003e\n\u003cp\u003eFinally, PERMANOVA allowed to summarize the pattern of plant and AM fungal parameters and to highlight significant interactions between Y, G and Inoc (Table S4). The PCO biplot (Fig. 6) showed that all genotypes differently responded to inoculation in the two year of cultivation. Although the year of cultivation was very strongly affecting the pattern of reponse, with an explained variance of 65%, the third-order interaction explained 7%. Looking at the PCO biplot, it showed a clear difference between years. In 2021 the agronomic reponse was less variable among replicate plots, and this encouraged a further investigation on root AM fungal communities. Our results are in contrast with our first hypothesis that genotype exerts a greater control over the response of barley to AM fungal inoculation than the environment. Indeed, our hypothesis was based on recent findings that wheat genotype inoculated in the field with the same indigenous consortium was a major driver of the agronomic response (Marrassini et al. 2024). This supports a less variable response of wheat. \u003c/p\u003e\n\u003cp\u003eThe PERMANOVA pairwise comparisons, utilised to dissect this interaction, highlighted consistent differences among the inoculated and not inoculated group of varieties and in 2020 and in 2021 (Table S5). Concerto was consistently and positively affected by AM fungal inoculation in both years, while Atomo and Atlante only in 2020 and 2021, respectively. This supports a robust and stable response of Concerto to inoculation across years and demostrates its less susceptibility to pedo-climatic variability. Therefore, the modern crossbreed variety Concerto developed in UK, showed a high mycorrhizal responsiveness to the indigenous AMF and this highlight the a good local adaptation of the crop to soil and AMF, regardless their origin. Furthermore, the year was consistently found to significantly affect the pattern of plant and AM fungal parameters in both inoculated and not-inoculated groups of barley varieties, underlining the major role of environment in shaping plant response under the same agronomic practice. Finally, PERMDISP showed significant difference among barley genotypes (Table S4). A higher variable pattern of response was observed in Atomo, whereas less variable patterns in Concerto and Atlante (Fig. 6). Therefore, irrespective to inoculation and year of cultivation, the response of Concerto and Atlante are more stable. \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e3.3. Diversity of AMF \u003c/strong\u003e\u003cstrong\u003ein the roots of barley\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo test our hypothesis that host plant preference in the AMF colonizing plant genotypes inoculated with indigenous AMF is driven by changes of community structure and not by changes in composition of AMF, we investigated the AM fungal community diversity in roots of the three barley genotypes. The study allowed also to investigate host plant preference under no inoculation. Moreover, we investigated the role of plant growth stage on modulating host plant preference. We focused the investigation on root samples collected in 2021.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e3.3.1. Illumina sequencing output, AM fungal richness and alpha-diversity in barley roots\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAfter curation of the AM fungal sequences, 54,214 reads, ranging from 535 to 2,507 reads per sample, were retrieved and assigned to 26 VTXs (Fig. S2; Table S6). The 26 AMF VTXs belonged to three orders (i.e., Diversisporales, Entrophosporales, Glomerales) and four families (i.e., Diversisporacea, Gigasporaceae, Entrophosporaceae, Glomeraceae). The accumulation curves and rarefaction analyses of AMF confirmed that the Illumina sequencing effort was sufficient for the analysis (data not shown).\u003cem\u003e \u003c/em\u003eBarley genotype and inoculation differently affected AM fungal richness (S) and alpha-diversity (H\u0026rsquo; and \u0026lambda;) in the two growth stages (G12 and GS90) (Table S7). Atlante showed a higher diversity (S and \u003cem\u003eH\u003c/em\u003e\u0026rsquo;) than the other genotypes (+51% and +22%, respectively) (Table S8). \u003c/p\u003e\n\u003cp\u003ePreviously, the AM fungal diversity was only studied in single varieties of barley utilizing the taxonomic-based assessment of spores in soil and the molecular characterization in roots (Aguilera et al., 2017; Kaidzu et al. 2020). Therefore, our study highlights for the first time a host plant preference in AMF across barley genotypes. This is in accordance with the results obtained among genotypes of other crops (Kavadia et al. 2020; Mao et al. 2014; Parvin et al. 2021). Indeed, root traits variability among varieties of cereals, including barley, can be large (Nakhforrosh et al., 2014; Sendek et al. 2019; Robinson et al. 2018). Therefore, differences in root architecture could have determined the observed variable pattern of AM fungal diversity. \u003c/p\u003e\n\u003cp\u003eHowever, Inoc increased AM fungal richness and \u003cem\u003eH\u003c/em\u003e\u0026rsquo; only in Atlante at GS14 (+133% and +75%, respectively), while \u0026lambda; was promoted in Atlante and Atomo only at GS14 (+54% and +68%). In addition, AM fungal richness was higher at GS90 than GS14 (+23%). Since AM fungal diversity in roots promote plant productivity by the promotion of the abundance of productivity-promoting AMF (van der Heijden et al. 1998; Vogelsang et al. 2006), when inoculation determines the promotion of the diversity traits (i.e., Atlante and Atomo), a benefit of productivity can be expected.\u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e3.3.2. Community composition and structure of AMF \u003c/strong\u003e\u003cstrong\u003ein barley roots\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eSixty-five percentage of AM fungal taxa (VTXs) (65%) were shared among genotypes, irrespective to AM fungal inoculation and plant growth stage (GS). Similarly, barley genotypes shared 52% and 50% VTXs in inoculated conditions and in control, irrespective to GS. Moreover, at GS14 the percentage of VTXs shared in inoculated conditions was similar to the one in the controls (32% and in 37%), while at GS90 it reached 65% in inoculated plants and 40% in controls. These results support our hypothesis of a good host preference in AMF across genotypes of barley in both inoculation treatments. They are in accordance with the variability in AM fungal composition observed in field across three wheat genotypes cultivated in a similar climatic area (Marassini et al., 2024; Pellegrino et al. 2020). Thus, also in barley there is a core composition of AMF that can be considered generalists, but there are several specialist taxa uniquely retrieved in the roots of each genotype. Moreover, since inoculation with indigenous AMF did not modify the rate of shared taxa at early stage, we can state that locally-sourced inocula did not change the AM fungal recruitment of the crop. By contrast, the increase of shared VTXs observed at GS90 due to inoculation, allows to highlight that indigenous AM fungal inoculants can reduce the variability in host preference among genotypes. Similarly, inoculating the same AM fungal consortium in wheat, the percentange of shared VTXs at maturity was higher than in the controls (46% \u003cem\u003evs\u003c/em\u003e 35%) (Marrassini et al. 2024). This supports the idea that indigenous taxa contained in the inoculum, during crop growth, colonize a larger number of plant genoypes and that our consortium was composed by high-compatible taxa.\u003c/p\u003e\n\u003cp\u003eFocusing on the pattern of AM fungal composition in inoculated and not-inoculated barley genotypes, the average percentage of shared VTXs was 51% and 65% at GS14 and GS90, while the percentage of VTXs uniquelly retrieved in inoculated plants was 23% and 18%, respectively. Thus, we can not fully confirm our hypothesis that inoculation with a local AM fungal consortium did not modify the composition of the AM fungal communities. However, we can assume that when indigenous inoculants are applied they start to compete with closely related AM fungal species present in the soil at very low abundance, and thus not detectable by the molecular tools. Indeed, when individuals are rare in a microbial community, Illumina sequencing has some limitations in the accurancy of detection (Cheng et al. 2023; Egan et al. 2018). Moreover, the VTXs uniquelly found in the inoculated barley genotypes had a very low abundance (0.6%). \u003c/p\u003e\n\u003cp\u003eOur findings are in accordance with previous results obtained on bread wheat genotypes and in sunflower, inoculated with the same AM fungal consortium in similar climatic conditions (Arcidiacono et al. 2024; Marrassini et al. 2024). Therefore, our data supports the low environmental and ecological impact of local AM fungal inoculants. Thus, biofertilization with indigenous AMF should be considered a suitable practice for the management of cropping systems. \u003c/p\u003e\n\u003cp\u003eLooking at AM fungal community structures in the roots of barley genotypes in the PCO plot and in the shade plot (Fig. 7a,b), we can observe the significant interaction between G, Inoc and GS. This is supported by the results of PERMANOVA that highlighted a high percentage of explained variance of the third order interaction (24%) (Table S9). Overall, at GS14, no differences were recorded among not-inoculated barley genotypes, while significant differences were recorded between inoculated Atlante and Atomo/Concerto (Table S10). At GS90, no differences were recorded among not-inoculated barley genotypes and neither among inoculated genotypes. Therefore, at early and late plant development and under no inoculation, host preference in AMF is more discriminated by the pattern of composition than by the pattern of the community structure. The same occured at plant maturity in inoculated genotypes. By contrast, at early plant development, in inoculated conditions, host preference is discriminated by both composition and community structure. These findings highlight that to study host preference in managed field crops it is important to take into account these two aspects during the growing season. \u003c/p\u003e\n\u003cp\u003eMoreover, inoculation determined at GS14 changes in AM fungal community structure only in Atlante, while at GS90 in both Atlante and Atomo. This results support the high variability in AM fungal community structure among wheat genotypes (Mao et al. 2014; Marrassini et al., 2024; Pellegrino et al. 2020) and gave new insights in the mechanisms by which diversity within AM fungal populations is enhance or maintain in roots. Indeed, a genotype like Concerto whose AM fungal community structure was similar in inoculated and not-inoculated treatments and AM fungal abundace and yield were promoted, can be considered a suitable variety in biofertilization programs. Previously, other factors, such as soil phosphate availability and host species were identified as main determinant of the root AM fungal structure (Cavagnaro et al., 2005; Ehinger et al., 2009; Eom et al. 2000; Helgason et al. 2002; Vanderkoornhuyse et al. 2003). Finally, the fact that two barley genotypes, Atomo and Atlante, in not-inoculated and inoculated conditions, respectively, showed significant differences in the AM fungal community structure between growth stages, is in accordance with a previous work reporting differences along the plant cycle in bread wheat, oat and barley, inoculated and not with a AMF (Aguilera et al. 2021). The importance of the phenological stage in shaping AM fungal community structure in roots of wheat was also reported in field conditions (Marrassini et al. 2024) and and perennial and annual grasses and non-grasses species (Lingfei et al. 2005). The role of barley phenological stage was underlined also by PERMDISP (Table S9). The higher variability at the early development of barley can be attributed to mechanisms of competition among AMF within a community (Chagnon et al., 2013; Jansa et al. 2008;). \u003c/p\u003e\n\n\u003cp\u003e\u003cstrong\u003e3.4. Modelling barley productivity against AM fungal abundance and structure in roots\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eOur results showed that there was a significant relationship between the traits of AM fungal abundance in roots and barley productivity (RELATE: \u0026rho;=0.587; \u003cem\u003eP\u003c/em\u003e=0.001) (Fig. 8) and the BEST analysis allowed us to highlight arbuscules as the best predictor of barley productivity. Therefore, as we hypothesized, under low soil fertility AM fungal colonization traits, such as the percentage of arbuscules, are key and consistent determinants of the productivity of barley. Similarly, in field conditions, arbuscules were positively and strongly correlated with all functional traits of sunflower grown at low and high soil fertility (Arcidiacono et al. 2024). The association between increased AM fungal root colonization and increased barley productivity is consistent with the results of meta-analytic works (Lekberg and Koide 2005; McGonigle 1988; Pellegrino et al., 2015; Treseder, 2013).\u003c/p\u003e\n\u003cp\u003eMoreover, the agronomic response of barley was driven also by changes in the structure of the AM fungal community, as highlighted in Fig. S3 and by the significance of RELATE analysis (\u0026rho;=0.514; \u003cem\u003eP\u003c/em\u003e=0.005) (Fig. 8c), and not by changes of composition (\u0026rho;=0.084; \u003cem\u003eP\u003c/em\u003e=0.167; data not shown). This relationship is clear comparing the pattern of AM fungal community structure in roots of barley genotypes at GS90 displayed by the nMDS plot (Fig. S3a) and the pattern of yield and nutrient uptake displayed by the PCO biplot (Fig S. 3b). Therefore, we confirmed our hypothesis that changes in AM fungal community structures and not in composition determine barley productivity. Moreover, the BEST analysis highlighted the best predictors of barley productivity (\u0026rho;=0.44, \u003cem\u003eP\u003c/em\u003e=0.046) that was \u003cem\u003eGlomus\u003c/em\u003e sp. VTX00342, considering one descriptor (r=0.333), or the taxa VTX00342 with \u003cem\u003eSeptoglomus \u003c/em\u003esp. VTX00064, considering two descriptors (r=0.406) (Fig. 8d). These BEST predictors were also highlighted in the nMDS plot (Fig. S3a). Indeed, \u003cem\u003eGlomus\u003c/em\u003e sp. VTX00342 and \u003cem\u003eSeptoglomus \u003c/em\u003esp. VTX00064, putative members of the indigenous AM fungal inoculum, were more present in inoculated than not-inoculated plots (+46% and +56%, respectively). Previously, changes in AM fungal community structure and not in composition were identified as main driver of crop productivity under inoculation with indigenous AMF (Arcidiacono et al., 2024; Marrassini et al., 2024; Pellegrino et al. 2022). Indeed, the inoculated indigenous strains, highly compatible with all barley genotypes, as well as with the local environmental conditions, are preferentially recruited by the roots. \u003c/p\u003e"},{"header":"Conclusions","content":"\u003cp\u003eThe general positive outcome in barley productivity supports the use of local AMF for building future inoculant generations and their large inclusion in sustainable agriculture. These results are particularly important under the current context where most minerals are significantly decreased in grain due to the dilution effects caused by intensification of agricultural management and high-yielding plant breeding. However, the underlined effect of environment in modulating the response of barley can help in the selection of genotypes with stable AM fungal response in specific climatic conditions. This is of key importance given the current challenges related to climate change. Moreover, our results better diclose the mechanisms at the basis of the host preference. Therefore, we suggest the propagation of AM fungal consortia adapted to specific climatic contexts to boost AM fungal inoculum production and to reduce the potential ecological risks of the exotic AM fungal inoculants.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u0026nbsp;\u003c/strong\u003eThe authors thank Domenico D\u0026rsquo;Alessio for hosting the field experiments on the farm Rinnovamento Agricolo, Santa Luce, Pisa (Italy). We also thank Dr. Giusto Giovannetti of CCS Aosta for large-scale culturing of the AMF inoculum.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u0026nbsp;\u003c/strong\u003eThis work was funded by the European Agricultural Fund for Rural Development 2007-2013 for Tuscany (Italy) measure 16.1 (FERTIBIO project; CUP Artea 743548; project leader Dr. Elisa Pellegrino) and measure 16.2 (FERTIBIO project; CUP Artea 828090; project leader Prof. Laura Ercoli. Valentina Marrassini was supported by a PhD scholarship in Agrobiosciences (XXXVI cycle) from the Scuola Superiore Sant\u0026rsquo;Anna (SSSA, Pisa, Italy).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eDeclaration of competing interest\u0026nbsp;\u003c/strong\u003eThe authors declare that they have no known competing financial interests or personal relationships that could have influenced the work reported in this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent to participate\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u003c/strong\u003e All the authors whose names appeared on the submission approved the version to be published and agreed to be accountable for all aspects of the work in ensuring that the questions related to the accuracy of integrity of any part of the work were appropriately investigated and resolved.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u003c/strong\u003e Data will be made available on request. Furthermore, DNA sequences are available in the NCBI Sequence Read (SRA) database as SUB14254691.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCode availability\u003c/strong\u003e Not applicable.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor\u0026rsquo;s contributions\u003c/strong\u003e Conceptualization, supervision, methodology, resources and funding acquisition, LE and EP; methodology, investigation, formal analysis, VM; data curation, result interpretation, visualization, writing original draft, review, and editing: VM, EP; review and editing: AVAP, LE. All authors gave their final approval for publication.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eAguilera P, Mar\u0026iacute;n C, Oehl F, Godoy R, Borie F. Cornejo P (2017) Selection of aluminum tolerant cereal genotypes strongly influences the arbuscular mycorrhizal fungal communities in an acidic Andosol. 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New Phytol 222 (1): 543-555. https://doi.org/10.1111/nph.15570\u003c/li\u003e\n\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":true,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"agronomy-for-sustainable-development","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ASDE","sideBox":"Learn more about [Agronomy for Sustainable Development](https://www.springer.com/journal/13593)","snPcode":"13593","submissionUrl":"https://www2.cloud.editorialmanager.com/asde/default2.aspx","title":"Agronomy for Sustainable Development","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false},"keywords":"native arbuscular mycorrhizal fungi, barley genotypes, barley nutrient uptake, mycorrhizal yield benefit, molecular diversity, field inoculation","lastPublishedDoi":"10.21203/rs.3.rs-4314201/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-4314201/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"In barley cultivation, high use of mineral fertilisers in combination with low crop nutrient use efficiency results in severe environmental and economic issues. In this context, inoculants with indigenous arbuscular mycorrhizal fungi (AMF) could represent an efficient solution where intensive agriculture negatively impacted soil AM fungal abundance and diversity. However, since crop breeding and environment can strongly affect plant mycorrhizal response, in this work, we tested the agro-ecological effect of field inoculation with a indifìgenous AM fungal consortium on three varieties of barley for two years. In 2020, when soil was clay loam with very low P availability and no drought stress, Atomo and Concerto varieties positively responded to inoculation in terms of AM fungal traits, whereas in 2021, with silty clay loam soil, low P availability and drough stress, only Concerto was responsive. In 2020, inoculation promoted grain yield by 64% and 37% in Atomo and Concerto, and in 2021 by 78% and 134% in Concerto and Atlante. Multivariate analysis highlighted a strong effect of environment on barley productivity and a third-order significant interaction AMF, genotype and environment (65% and 7% of explained variance). Inoculation slightly modified AM fungal composition, it strongly modified, together with plant growth stage, the AM fungal community structures. A significant relationship between root AM fungal abundance and barley productivity was highlighted, with arbuscules as best predictor. Accordingly, changes in AM fungal root community structure and not in composition drove barley response and the main players were Glomus sp. VTX00342 and Septoglomus sp. VTX00064, putative members of the local AM inoculum. The general positive barley productivity outcome supports the use of indigenous AMF for building efficient and ecologically safe inoculants and their inclusion in sustainable agriculture. Nevertheless, the selection of genotypes with stable AM fungal response in specific climatic conditions is crucial in biofertilization programmes.","manuscriptTitle":"Positive response of barley to an indigenous arbuscular mycorrhizal fungal (AMF) inoculant is modulated by genotype and environment through changes in AMF root abundance and community structure","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-07-18 05:59:12","doi":"10.21203/rs.3.rs-4314201/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"reviewerAgreed","content":"","date":"2024-07-11T19:50:37+00:00","index":0,"fulltext":""},{"type":"reviewersInvited","content":"","date":"2024-06-26T13:23:05+00:00","index":"","fulltext":""},{"type":"editorInvited","content":"Agronomy for Sustainable Development","date":"2024-05-06T10:00:36+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2024-04-29T09:50:03+00:00","index":"","fulltext":""},{"type":"submitted","content":"Agronomy for Sustainable Development","date":"2024-04-23T16:23:14+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"agronomy-for-sustainable-development","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"ASDE","sideBox":"Learn more about [Agronomy for Sustainable Development](https://www.springer.com/journal/13593)","snPcode":"13593","submissionUrl":"https://www2.cloud.editorialmanager.com/asde/default2.aspx","title":"Agronomy for Sustainable Development","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"em","reportingPortfolio":"Springer Hybrid","inReviewEnabled":true,"inReviewRevisionsEnabled":false}}],"origin":"","ownerIdentity":"2bda3c65-28cf-4ce5-aed5-3ede73f923db","owner":[],"postedDate":"July 18th, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-02-27T12:19:03+00:00","versionOfRecord":[],"versionCreatedAt":"2024-07-18 05:59:12","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-4314201","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4314201","identity":"rs-4314201","version":["v1"]},"buildId":"qtupq5eGEP_6zYnWcrvyt","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}