Introduction

In recent years, the rising demand for the production of environmentally safe and high-quality food has caused a major shift in agricultural management, which is growing increasingly sustainable by making use of practices able to maintain and enhance soil fertility and health (FAO 2011). In this context, soil microorganisms play a key role in the modulation of soil biochemical, biological, and nutritional processes (Azcón-Aguilar and Barea 2015). Such microbiota thrives in a privileged niche at the soil–root interface (rhizosphere) that is a rich source of nourishment, represented by sugars, amino acids, and organic acids in the form of root exudates (Philippot et al. 2013). The complex microbial communities establishing in the rhizosphere have profound effects on plant growth, nutrition, and health (Compant et al. 2010; Hayat et al. 2010). Members of the rhizospheric microbiota may establish an intimate relationship with their host plants, colonizing roots and also aboveground plant compartments, becoming endophytes, i.e., microorganisms which can be isolated from, or detected within, surface-sterilized plant organs and do not cause visible harm to the host organism (Hardoim et al. 2008). Bacterial endophytes of plant roots may reach a density of 104–108 bacterial cells per gram of root tissue and may have important roles in plant growth promotion, fitness, and protection against pathogens. In exchange, the plant endosphere provides the endophytic microbiota with a more uniform, protected, and nutrient-rich environment than the nearby soil (Hardoim et al. 2015; Liu et al. 2017b). The use of green fluorescent protein labeling, image analysis, and fluorescence in situ hybridization–confocal laser scanning microscopy has allowed the localization of bacterial endophytes in intercellular spaces, at the base of lateral roots and root tips, in xylem vessels, and inside root hairs (Compant et al. 2010; Gaiero et al. 2013; Liu et al. 2017a).

Arbuscular mycorrhizal fungi (AMF) represent an important group of root endophytes in view of their key role in the enhancement of plant nutrition, health, and product quality. AMF (phylum Glomeromycota) are beneficial soil fungi, establishing mutualistic symbioses with about 80% of land plants including the major food crops, such as cereals, pulses, vegetables, and fruit trees and industrial crops like cotton, sunflower, and oil palm (Smith and Read 2008). They are obligate biotrophic organisms which obtain carbon from the host plant, providing in exchange soil mineral nutrients (such as P, N, S, K, Ca, Cu, and Zn), absorbed and translocated by the extraradical mycelium (ERM) extending from colonized roots into the soil. Therefore, the ERM represents an efficient auxiliary absorbing system because of its high surface-to-volume ratio, hyphal P absorption beyond the P depletion zone around roots, and the occurrence of many nutrient transporters in its hyphae (Smith and Read 2008; Pepe et al. 2017; Kameoka et al. 2019). Moreover, AMF improve plant performance and health by increasing plant tolerance to biotic and abiotic stresses (Sikes et al. 2009; Bitterlich et al. 2018) and induce changes in plant secondary metabolism leading to enhanced biosynthesis of health-promoting phytochemicals (Avio et al. 2018; Agnolucci et al. 2020). Overall, AMF provide multifunctional ecosystem services and are utilized as biofertilizers, biostimulants, and bioenhancers in agriculture (Gianinazzi et al. 2010; Rouphael et al. 2015).

Arbuscular mycorrhizal fungi live closely associated with large and diverse bacterial communities which may colonize spores, sporocarps, and extraradical hyphae, originating a complex and metabolically active environment called the mycorrhizosphere (Rambelli 1973). Such microbiota show different plant growth–promoting properties (plant hormone, antibiotic, and siderophore production; N2 fixation; P solubilization) and mycorrhiza helper activities (spore germination and mycelial growth promotion, mycorrhizal establishment facilitation) also have been observed, leading to enhancement of host plant performance (Bharadwaj et al. 2008a; Battini et al. 2016; 2017; Sharma et al. 2020). The potential functional complementarity and synergistic activity of AMF and their associated microbiota necessitate studies aimed at understanding the complex network of interactions between them and their host plants (Turrini et al. 2018). This is all the more important when implementing AMF inocula in sustainable and organic agriculture where the functioning of such multipartite associations may be crucial for crop production. Notwithstanding, scanty information is available on the relationship between AMF-associated bacteria and the bacterial microbiota colonizing roots after AMF inoculation. Here, we (i) provide an overview of recent developments regarding the recruitment of root endophytic bacteria, (ii) present data on the diversity and functionality of AMF-associated bacterial communities, and (iii) discuss the possible role of AMF in shaping the structure and composition of endophytic bacterial communities recruited by plant roots.

Endophytic bacteria recruited by plant roots

Characteristics and importance of root endophytic bacteria

Bacterial endophytes are able to recognize plant root exudates, adhere to the root surface, form a biofilm, and then enter roots, colonizing their inner tissues. According to current knowledge, passive penetration can take place at wounds, root cracks, secondary root emergence points, and root tips, while active colonization can involve cell wall–degrading hydrolytic enzymes (Compant et al. 2010). The presence of flagella, pili, lipopolysaccharides, exopolysaccharides, some special membrane proteins, quorum-sensing signals, chemotactic abilities, protein secretion systems, and twitching motility also may have importance in the invasion processes of certain endophytic bacteria, as shown by comparative genome analyses (Sessitsch et al. 2012; Hardoim et al. 2015; Pinski et al. 2019). Some of the required endophytic/symbiotic genes may be coded on plasmids or “symbiosis islands,” suggesting the possibility of horizontal transfer of these functional genes among the members of soil bacterial communities (Finan 2002). Nevertheless, vertical ways of endophyte transmission (seed-borne endophytes) also have been confirmed (Truyens et al. 2015) in which cases the transmitted bacteria were able to colonize the rhizospheres of new plantlets (Kaga et al. 2009; Hameed et al. 2015).

Bacteria colonizing the root endosphere profit from enhanced nutrient availability and environmental homeostasis provided by the plant, while the host plant may receive benefits from the endophytes in the form of direct and indirect growth promotion, stress reduction, or inhibition of plant pathogens (Gaiero et al. 2013; Hardoim et al. 2008; 2015; Compant et al. 2010; Pinski et al. 2019). As to nutrient mobilization, many endophytic bacteria were reported to possess N2-fixing, nitrifying, denitrifying, and P-solubilizing abilities or to be able to produce siderophores (Sessitsch et al. 2012; Hameed et al. 2015). Bacterial root endophytes influence plant hormone levels directly or indirectly, affecting growth, stress, and immune responses of host plants. Many endophytes may be able to release indoleacetic acids (IAA), gibberellins, cytokinins, ethylene, abscisic acid, jasmonates, and volatile compounds, while some of them also act as a sink for 1-aminocyclopropane-1-carboxylate (ACC), a precursor of ethylene production, because of a cytoplasmic ACC deaminase enzyme. The features mentioned suggest that bacterial endophytes may have a fundamental role in “fine-tuning” the hormonal balance of the host plant (Forchetti et al. 2007; Glick 2014; Hardoim et al. 2015). Furthermore, a key feature of endophytic strains is detoxification of reactive oxygen species, reactive nitrogen species, glutathione synthases, and glutathione-S-transferases, ameliorating different effectors of plant stress responses (Sessitsch et al. 2012). In the unique ecological niche where they thrive, some root endophytic bacteria may produce bioactive secondary metabolites, such as antibiotic and antiviral compounds (Strobel et al. 2004; Ryan et al. 2008; Ek-Ramos et al. 2019), while others have shown potential for enhancement of phytoremediation procedures because of their ability to decompose contaminants (Ryan et al. 2008; Mitter et al. 2019). Some authors have reported that the root microbiota may contribute to improved food quality through biofortification or production of health-promoting metabolites (Rehman et al. 2018; Ku et al. 2019) and potentially affect the quality of processed food products. For example, Minervini et al. (2015) demonstrated that endophytic lactic acid bacteria occurred not only in roots and various organs of durum wheat plants (Triticum turgidum ssp. durum) but also in the flour, possibly inducing changes in microbial community structure and properties of sourdough and derived products. Accordingly, the isolation and selection of functional root endophytic bacterial strains may be of particular interest for agriculture, industrial biotechnology, and medicine.

Recruitment of bacterial root endophytes by host plants

The quantitative and qualitative composition of root endophytic bacterial communities is influenced by many factors, mainly by geographical location, soil source, host genotype, and cultivation practice (Edwards et al. 2015). In Table 1, data from recent metagenomic studies of endophytic bacteria are reported. During notable work of defining the core root microbiome of the non-mycorrhizal species Arabidopsis thaliana L., based on 16S rDNA high-throughput sequencing data, two research groups revealed that the composition of root endophytic bacterial communities may be influenced by host genotype (Bulgarelli et al. 2012; Lundberg et al. 2012). Other studies demonstrated that the host plant may modulate the occurrence of root microbiota recruited from the nearby soil environment. Manter et al. (2010), utilizing a pyrosequencing approach, investigated the root endophytic communities of 20 potato cultivars and clones, revealing significant differences among the taxonomic profiles within those different plant genotypes. In that study, the identified bacterial operational taxonomic units (OTUs) affiliated with 238 genera and 15 phyla, demonstrating the remarkable diversity and variability of root endophytes. Plant genotype was found to shape the community composition of bacteria associated with the roots of 10 different rice cultivars (Hardoim et al. 2011), while differences in the bacterial root microbiota in the non-mycotrophic Brassicaceae family were found to be largely quantitative (Schlaeppi et al. 2014). Using the Illumina MiSeq platform, Marasco et al. (2018) found that grapevine (Vitis vinifera L.) rootstock genotypes influenced the taxonomic composition of their endophytic bacterial communities, although plant growth–promoting traits were not significantly different among the cultivars, showing a homeostasis of the plant/bacterial endophyte relationship. Culture-independent techniques, PCR-denaturing gradient gel electrophoresis (DGGE) and Illumina MiSeq sequencing of the 16S rDNA of root endophytic bacterial communities confirmed the selectivity of genotypes in durum wheat, as different cultivars hosted significantly different bacterial communities in their root tissues (Agnolucci et al. 2019b). However, Singer et al. (2019) found conserved community structures across different genotypes of Panicum virgatum and Panicum hallii.

Table 1 Relative abundance of the most represented bacterial phyla in the root endophytic communities of host plants from different ecosystems and geographic locations, as assessed by metagenomic approaches. In each study, only bacterial phyla with a relative abundance ≥ 2% were considered
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Not only the genotype, but also the phenological stage of the host plant may cause variability in the composition of root endophytic bacterial communities (Van Overbeek and Van Elsas 2008). Such an effect was clearly demonstrated in field-grown durum wheat, where bacterial taxa affiliated with Firmicutes showed fluctuating relative abundance in roots and other plant organs during the growing season (Minervini et al. 2015). Likewise, endophytic bacterial community compositions changed significantly across the growing stages in roots of sweet potato (Marques et al. 2015) and of non-mycorrhizal sugar beet (Shi et al. 2014).

Additional studies have revealed significant shifts in the composition and function of root bacterial microbiota as an effect of environmental variability. Soil type and geographic location contribute variability to potential soilborne colonizers causing significant differences in the quantitative and qualitative composition of root endophytic bacterial communities (Conn and Franco 2004; Lundberg et al. 2012; Schlaeppi et al. 2014; Edwards et al. 2015; Hameed et al. 2015). Other abiotic factors, such as stress (Naylor et al. 2017), flooding (Ferrando and Scavino 2015), suboptimal mineral nutrition (Hameed et al. 2015), seasons (Mocali et al. 2003), or agricultural management practices (Seghers et al. 2004) also have been identified as drivers of root endophytic bacterial community changes.

Regarding the taxonomic position of members of root bacterial microbiota, Liu et al. (2017b) reviewed previously published datasets, revealing that the main phyla were represented by Proteobacteria (ca. 50% in relative abundance), Actinobacteria (ca. 10%), Firmicutes (ca. 10%), and Bacteroidetes (ca. 10%). They also reported that Chloroflexi, Cyanobacteria, Planctomycetes, Verrucomicrobia, Nitrospirae, and Armatimonadetes were common in root tissues, while others, for example, Acidobacteria and Gemmatimonadetes, almost were excluded from the root endosphere. As previously mentioned, however, several studies have confirmed the active roles of host plants in the recruitment of selected bacteria from the nearby soil environment. For example, the genera Enterobacter, Pseudomonas, and Stenotrophomonas (Proteobacteria) represented the core bacterial endophytes in the roots of sweet potato and rice (Marques et al. 2015; Sessitsch et al. 2012). Accordingly, Pseudomonas-like OTUs dominated in the roots of Populus deltoides (34%) (Gottel et al. 2011), while Proteobacteria, Actinobacteria, and Bacteroidetes were the dominating phyla in root bacterial communities of A. thaliana (Bulgarelli et al. 2012; Lundberg et al. 2012; Bodenhausen et al. 2013), wheat, and tomato (Liu et al. 2017a; Lee et al. 2019). In addition to such taxa, Firmicutes occurred in the roots of Aloe vera and Capsicum annuum (Akinsanya et al. 2015; Barraza et al. 2020). Overall, the strong selection by the host plant results in recruitment of an endophytic bacterial community much simpler than that of the rhizosphere and of the nearby soil environment (Novello et al. 2017). 

Diversity and functionality of AMF-associated bacterial communities

The services provided by AMF often are facilitated by the large and diverse beneficial bacterial communities living closely associated with spores, sporocarps, and extraradical mycelium, frequently embedded in spore wall layers and, in sporocarpic species, in the microniches formed by peridial hyphae (Walley and Germida 1996; Filippi et al. 1998; Iffis et al. 2014) (Table 2). The mycorrhizosphere microbiota show diverse functional activities, ranging from the role of “mycorrhiza helper” (MH) to that of “plant growth promoter” (PGP). MH bacteria (MHB) can promote spore germination, mycelial growth, and mycorrhiza establishment, while PGP bacteria (PGPB) have the ability to enhance plant growth, nutrition, health, and stress resistance (Barea et al. 2002; Frey-Klett et al. 2007). In addition, some components of such beneficial microbiota possess both MH and PGP traits (Xavier and Germida 2003; Battini et al. 2017). Overall, MHB and PGPB show activities promoting and complementing those of AMF (Turrini et al. 2018; Giovannini et al. 2020). The metabolic traits underlying MH functions include growth factor production and detoxification of antagonistic substances, while PGP properties can range from N2 fixation, nutrient mobilization, and nutrient uptake facilitation to plant hormone, antibiotic, and siderophore production, or systemic resistance induction (Frey-Klett et al. 2007; Hayat et al. 2010).

Table 2 Bacterial communities closely associated with spores, sporocarps, extraradical mycelium and intraradical propagules of AMF species and isolates from different geographic locations
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From a taxonomic viewpoint, the composition of AMF-associated bacterial microbiota strongly depends on AMF identity. Indeed, PCR-DGGE analysis of 16S rDNA showed that the bacterial communities associated with AMF spores were more influenced by fungal than host plant species. Overall, PCR-DGGE allowed the detection of bacterial sequences affiliated with the genera Cellvibrio, Chondromyces, Lysobacter, Pseudomonas (Proteobacteria), and Flexibacter (Bacteroidetes). Such bacteria, in particular the genus Flexibacter, well known for their ability to degrade biopolymers, were suspected of feeding on the spore wall, which consists mainly of chitin (Roesti et al. 2005). The same molecular approach revealed differences in the composition of spore-associated bacterial communities of two AMF, Gigaspora margarita and Gigaspora rosea, and showed that most of the bacterial sequences from G. margarita were affiliated with Proteobacteria (Azospirillum, Azovibrio, Polyangium, Ramlibacter, Rubrivivax, Sphingomonas, Rhizobium) and Actinobacteria (Streptomyces, Amycolatopsis, and Pseudonocardia) (Long et al. 2008). Interestingly, by PCR-DGGE and band sequencing, Agnolucci et al. (2015) revealed that the spores of six different AMF harbored unique bacterial communities, which were not correlated with the taxonomic positions of the fungi. The sequences were affiliated with Actinomycetales, Bacillales, Burkholderiales, Pseudomonadales, and Rhizobiales, all orders encompassing taxa known as PGP bacteria. These three mentioned works reached consistent conclusions, suggesting that the differences in the composition of spore walls or spore exudates may affect the recruitment of spore-associated bacterial communities. In contrast, bacterial communities closely associated with AMF spores were reported to be shaped not only by fungal identity, but also by the identity of the host plant (Iffis et al. 2016). Gammaproteobacteria were more abundant in spores collected from Solidago canadensis soil samples than from Populus balsamifera and Lycopus europaeus, whereas spores belonging to the genus Glomus were correlated with Betaproteobacteria, Actinobacteria, Bacilli, and Sphingobacteria. In this case, the authors suggested that the strategy of differential bacteria recruitment by diverse AMF species and isolates also might reflect variations in the composition of spores/hyphae exudates, attracting specific microbial communities (Iffis et al. 2016). The underlying mechanisms of such differential recruitment among different AMF remain to be thoroughly investigated.

A novel study reported a surprisingly high diversity of bacteria associated with AMF vesicles and intraradical spores extracted from microdissected roots of Solidago rugosa. The dominant sequences belonged to the genera Sphingomonas, Pseudomonas, Massilia, and Methylobacterium (Proteobacteria) while Bradyrhizobium, Bosea (Proteobacteria), Bacillus, and Paenibacillus (Firmicutes) were found at lower frequencies (Iffis et al. 2014).

Only a limited number of studies have investigated the bacterial communities closely associated with the surface of AMF hyphae. One of the first works, by using bromodeoxyuridine immunocapture and confocal microscopy, determined the specific attachment of a strain of Bacillus cereus to AMF hyphae (Artursson and Jansson 2003). Toljander et al. (2006) reported that five different strains of gfp-tagged soil bacteria, inoculated into in vitro cultures of two Glomus isolates growing in a controlled artificial system – T-DNA-transformed roots – exhibited different levels of hyphal attachment. In particular, Paenibacillus brasiliensis, Bacillus cereus, Paenibacillus peoriae (Firmicutes), and Pseudomonas fluorescens (Proteobacteria) attached to AMF hyphae, while Arthrobacter chlorophenolicus (Actinobacteria) did not. Such differences can be ascribed to AMF hyphal exudates that have been reported to affect the composition of bacterial communities (Toljander et al. 2007). Similar in vitro experimental systems showed that Streptomyces and members of the Oxalobacteraceae family, i.e., Duganella, Janthinobacterium, and Massilia, were specifically attached to the surface of AMF hyphae (Scheublin et al. 2010). Interestingly, 26 Burkholderia spp. strains and one Rhizobium miluonense strain were able to strongly attach to Rhizophagus irregularis hyphae and to solubilize phosphate (Taktek et al. 2015). Consistent data were obtained by a recent work reporting the isolation of 128 bacterial strains from the hyphae of Rhizoglomus irregulare (syn. Rhizophagus irregularis), of which 12 showed phosphate-solubilizing activity (Sharma et al. 2020). A distinct bacterial community closely associated with extraradical hyphae of Glomus versiforme, and conserved across divergent soils, was mainly represented by Proteobacteria (50% relative abundance), Actinobacteria (10%), Chloroflexi (9%), Acidobacteria (7%), Bacteroidetes (6%), and Fibrobacteres (4%) (Emmett et al. 2021). An in vivo study reported, for the first time, that AMF hyphae may act as “transport agents” or “highways” for bacteria. Indeed, a gfp-tagged nitrogen-fixing rhizobial strain, Bradyrhizobium diazoefficiens, was able to tightly adhere to Glomus formosanum hyphae, facilitating bacterial translocation to their legume host plant and the formation of N-fixing nodules in the root system (de Novais et al. 2020). Recently, Illumina MiSeq metagenome sequencing allowed the identification of 276 bacterial genera, belonging to 165 families, 107 orders, and 23 phyla, mostly represented by Proteobacteria, Bacteroidetes, and Actinobacteria, associated with Rhizoglomus irregulare commercial inoculum. Such richness and diversity are remarkable, given that no bacteria were deliberately added to the AM symbiont. It is interesting to note that the predominant bacterial taxa correspond to those recurrently found in the root endosphere of the majority of plant species investigated so far (Table 1).

Culture-dependent analyses not only confirmed the high diversity of bacterial communities living in association with AMF, but also showed the PGP activity of strains belonging to Actinomycetales, Bacillales, Enterobacteriales, and Rhizobiales, as IAA and siderophore producers (Agnolucci et al. 2019a). Such activities are important for plant development because the phytohormone IAA is able to modulate the growth and functioning of the root system (Duca et al. 2014), while siderophores, high-affinity iron-chelating compounds, may facilitate plant iron acquisition and control soilborne diseases by means of iron competition (Mimmo et al. 2014). Other culture-based studies reported a high abundance of the mentioned phyla, recording genera such as Micrococcus, Acidovorax, Cellulomonas, Janthinobacterium, Alcaligenes, and Flavobacterium (Xavier and Germida 2003; Bharadwaj et al. 2008b; Cruz et al. 2008). Some bacteria with PGP potentials, isolated from the spores of Rhizophagus intraradices, affiliated with the genera Sinorhizobium/Ensifer, Streptomyces, Bacillus, Arthrobacter, and Fictibacillus, also have shown MH properties (Battini et al. 2016). Indeed, seven of such bacterial isolates significantly increased hyphal length density, while two of them, Streptomyces sp. W77 and Streptomyces sp. W94, additionally were able to promote specific P uptake and translocation in maize plants (Battini et al. 2017).

Overall, the available data show that diverse bacterial taxa are differentially able to attach to AMF structures, i.e., spores, sporocarps, and hyphae, of which exudates might differ in quantity and/or quality, thus promoting or inhibiting the growth and attachment of particular bacterial communities. It is important to note that such specific and close physical relationships may be indicative of complex interactions among AMF, bacteria, and host plants, suggesting that AMF might act as carriers of the endophytic microbiota that establish in roots.

Concluding observations and future prospects

The role of AMF as drivers of the endophytic bacterial communities colonizing plant roots can be revealed by investigating possible overlaps in the taxonomic composition of root endophytic microbiota and AMF-associated bacterial communities. Only a few such comparisons currently are available, but major shifts have been revealed in the composition of the root endophytic bacterial communities of durum wheat after inoculation with the AM symbiont Funneliformis mosseae. The use of two culture-independent approaches, PCR-DGGE analysis of 16S rDNA and high-throughput sequencing of the same gene through Illumina MiSeq, has revealed that AMF inoculation increased the abundance of some genera and species of Actinobacteria and Bacteroidetes in two durum wheat cultivars, Odisseo and Saragolla (Agnolucci et al. 2019b). In particular, Funneliformis mosseae increased the abundance of Actinobacteria, such as Rhodococcus species, in the cv. Saragolla, and that of Streptomyces and Microbacterium spp. in both cultivars. This is an interesting finding, as Actinobacteria, considered promising PGP bacteria (Seipke et al. 2012), have been reported to be closely associated with the spores of six different AMF isolates (Agnolucci et al. 2015). Moreover, in the two durum wheat cultivars, AMF inoculation increased the abundance of the Proteobacteria Pantoea, a PGP genus, including species and strains able to produce siderophores, enhance Zn availability in soil, and induce plant resistance to drought stress (Moreira et al. 2016; Kamran et al. 2017; Chen et al. 2017).

The possible mechanisms operating in the recruitment of different bacterial endophytic communities in roots may be dual. As the physiological status of plants is altered when they are mycorrhizal (e.g., photosynthesis, nutrition and growth, phenology, health) (Smith and Read 2008), certain groups of endophytic bacteria might find specific conditions best suited for their growth and development and thus might be favored in colonization of such a privileged ecological niche. On the other hand, they may gain facilitated access to root tissues during AMF establishment through their close association with AMF hyphae (Toljander et al. 2006; de Novais et al. 2020). These two mechanisms may act simultaneously, and it will be difficult to discriminate between them. Notwithstanding, systematic studies on the differential occurrence of root bacterial endophytes in mycorrhizal and non-mycorrhizal plants still are needed.

In the years to come, a series of key questions remain to be answered. The first question concerns whether members of AMF-associated bacterial communities can actually establish in the root system, becoming endophytic, and in what proportion. If so, would they be able to show the specific PGP properties revealed when isolated and grown in vitro? The results obtained will raise the question as to whether AMF identity and diversity can affect the structure and composition of root endophytic bacterial communities. Moreover, as AMF commercial inocula carry their own associated bacterial microbiota, could they represent suitable tools to introduce beneficial root endophytes in sustainable agriculture? Such questions can only be answered by targeted research on the diversity and functionality of root endophytic communities as affected by the presence of AMF and AMF-associated bacterial communities.