Introduction

Plants are hosts to complex communities of endophytic bacteria that colonize the internal of both below and above ground tissues without any apparent sign of infection or negative effects on the host plant [1]. These microorganisms can establish beneficial relationships with plants through direct and indirect mechanisms that enhance the availability and acquisition of nutrients, modulate plant hormone levels, induce pathogen resistance and mediate the quality of plant growth [2,3,4].

Chickpea (Cicer arietinum L.) is widely cultivated and currently ranks second in the world’s production of grain legumes [5]. It is a staple food worldwide valued as an important source of protein, carbohydrates, minerals and vitamins [6]. There is an increasing demand to implement biotechnological resources, including utilizing beneficial microorganisms, as an alternative to synthetic agrochemicals that have created serious environmental and health concerns to support sustainable crop growth and productivity.

The application of microbial inoculants, including rhizobia and other plant growth-promoting bacteria (PGPB) is a viable approach to improving chickpea growth and productivity, assisting plants in coping with environmental stresses and supporting sustainable agricultural systems [7]. However, the efficiency of beneficial microorganisms demonstrated in laboratory and greenhouse trials is not always consistent under field conditions. Therefore, the development and application of microbial inoculants from the plant core microbiome (microorganisms that are stably associated with one plant species, independent of soil physical and chemical characteristics and the environmental conditions and may form cores of interactions that can optimize microbial functions at the individual plant and ecosystem levels [8, 9] is suggested as a strategy to improve biofertilizers efficiency and ensure sustainable production patterns [10].

Nevertheless, despite its importance, interactions between plants and endophytic bacteria have not been studied extensively, except for rhizobia-legume symbiosis [2, 11]. Studies on the diversity of endophytic root bacteria revealed multiple genera associated with chickpea, with Pseudomonas, Pantoea, Enterobacter, Bacillus, Rhizobium, Microbacterium, Paenibacillus, Achromobacter, Mesorhizobium, Sphingomonas, and Burkholderia being the most common ones [11,12,13].

In addition to the root-associated endophytic bacterial community, there is an emerging view that plants can benefit from the seed microbiota, as its presence in the early stages of plant growth can assist germination and seedling establishment [14,15,16]. Besides, they may have a critical role in determining plant microbiota assembly, structure and function, and ultimately, plant productivity [14, 17, 18]. However, this link is still poorly understood, and the knowledge focused on the role of the seed-borne endophytic community in supporting the host plant growth has crucial gaps.

There is evidence that seed endophytes may spread as seeds disperse, and can be transmitted vertically via the seed from generation to generations, representing a widespread and ancient relationship in nature [14, 19,20,21]. Seed associated microorganisms are conserved across several plant generations, suggesting that plants may reduce the transmission of pathogenic symbionts to seeds, and plants that inherit symbionts with beneficial traits may have an ecological advantage in coping with environmental stresses through poorly understood mechanisms [14, 20].

Uncovering the seed microbial composition and function can lead to a better understanding of the plant as a meta-organism and how that impacts plant ecology, health, and productivity [8, 15]. This study aimed to isolate, identify and characterize culturable endophytic bacteria of chickpea seeds. We hypothesized that seeds are a reservoir of plant growth-promoting bacteria with potential for biotechnological and agronomic traits.

Material and Methods

Isolation of Culturable Endophytic Bacteria

Dry chickpea (Cicer arietinum L. cv. ‘Elixir’) seeds were obtained from plants inoculated with Pseudomonas sp. DSM 33,393 (MN880080), Burkholderia sp. DSM 33,394 (MN880078), Mesorhizobium sp. DSM 33,395 (MN880079), and Bacillus thuringiensis subsp. aizawai (TUREX) (commercial biopesticide). A detailed description of the bacteria identification, inoculation treatments and experimental site can be found in Laranjeira et al. [22]. Seeds (n = 200) were surface sterilized in 3% sodium hypochlorite for 3 min, followed by 70% ethanol for 1 min, and, subsequently, rinsed three times with sterile distilled water. Afterward, the seeds were aseptically macerated with glass rods and used to inoculate tryptic soy agar. After incubation at 28 °C for 72 h, a representative number of single colonies with different morphologies (based on size, shape and color) were picked up and sub-cultured separately until pure cultures were obtained. A total of 35 bacterial cultures were used for further analysis.

Identification and Phylogenetic Analysis of Endophytic Bacteria

Bacterial cells were lysed with CTAB and sterile glass. A mixture of phenol, chloroform and isoamyl alcohol (25:24:1) was added to the solution and then centrifuged (20 min at 13,000 rpm). The DNA was precipitated by adding 0.6% v/v isopropanol. The pellet was washed with 70% ethanol, dried and suspended in sterilized ultra-pure water. The 16S rRNA gene was amplified using sets of primers, fD1 and rD1 or 27F and 1492R [23, 24]. The PCR reaction was prepared as follows: 5.5 µl genomic DNA, 7.5 µl 2 × MyTaq™ HS Mix (Bioline Reagents Ltd United Kingdom) and 1 µl of each primer according to the manufacturer’s instructions. The amplification program used was: 3 min at 95 °C for initial denaturation; 34 cycles of 30 s at 95 °C, 30 s at 54 °C and 2 min at 72 °C; and a final extension for 10 min at 72 °C (Bio-Rad Thermocycler). PCR products were purified and sequenced by StabVida (Lisbon, Portugal).

The obtained sequences were compared for similarity to sequences deposited in GenBank hosted by the National Center for Biotechnology Information (NCBI), using the Basic Local Alignment Search Tool (BLAST) program. For phylogenetic analysis, sequences were aligned with the most similar sequences retrieved from the NCBI database using Multiple Alignment using Fast Fourier Transform (MAFTT). Maximum Likelihood (ML) phylogenetic trees were constructed in Molecular Evolutionary Genetics Analysis (MEGA) software version 6.0, using GTR + G + I (five categories) substitution model and considering all sites in datasets. Bootstrap support was based on 1000 replicates. Sequences generated in this study were deposited in the GenBank database under the following accession numbers: MW672282 to MW672316.

Plant Growth-Promoting Properties of Bacterial Endophytes

The isolates were tested for a range of plant growth promotion characteristics. Phosphate solubilization activity was determined using the National Botanical Research Institute’s phosphate growth medium (NBRIP) containing as a single phosphorus source, 5 g L−1 of tricalcium phosphate (TCP, Ca3(PO4)2) or aluminum phosphate (AlPO4) [25]. The isolates were incubated at 28 °C for 3 days. The formula ((colony + colored zone diameter)/colony diameter) was used to distinguish the isolate’s ability to solubilize phosphorus [26]. The same formula was used to assess siderophore production by the Chrome Azurol S assay [27] after incubation at 30 °C for 5 days. The ability of the isolates to produce hydrogen cyanide (HCN) was performed according to Bakker and Schippers [28]. Bacterial cultures were grown at 28 °C for 5 days on Tryptic Soy Agar supplemented with glycine (4.4 g L−1). Petri plates with upper lids contained a Whatman No.1 filter paper soaked in 0.5% picric acid and 2% sodium carbonate. A color change of the filter paper from orange to brown was considered an indication of HCN production. Indole acetic acid (IAA) production was performed according to Gordon and Weber [29] with some adaptations. Bacterial cultures were grown in Yeast Extract Mannitol supplemented with 250 µg ml−1 of tryptophan for 72 h at 28 °C. The cultures were centrifuged at 8 500 × g for 5 min, and 2 ml of supernatant was mixed with 4 ml of Salkowski’s reagent (1 ml 0.5 M FeCl3 solution in 50 ml of 35% HClO4) and 100 µl of orthophosphoric acid. After 25 min of incubation in the dark, the absorbance was measure at 530 nm. The concentration of IAA was calculated using a standard curve. In all experiments, three replications were maintained for each isolate.

Results

Identification of Bacterial Endophytes from Chickpea Seeds

The phylogenetic analysis based on the partial 16S rRNA nucleotide sequences revealed differences in the endophytic bacterial community composition. Thirty-five culturable bacterial endophytes were assigned to three phyla: Proteobacteria, Firmicutes, and Actinobacteria (Fig. 1). Firmicutes was the most abundant phylum, accounting for 65.7% of total isolates, belonging to the genera Bacillus, Priestia, and Paenibacillus. Within the Proteobacteria, isolates belonging to the classes Alphaproteobacteria (Mesorhizobium), Betaproteobacteria (Burkholderia and Alcaligenes), and Gammaproteobacteria (Acinetobacter, Rahnella, and Enterobacter) were found. Moreover, the genera Tsukamurella and Microbacterium were found among the Actinobacteria phylum.

Fig. 1
figure 1

Maximum likelihood tree based on the partial sequence of the 16S RNA gene of bacterial isolates from chickpea seeds and their related type strains. The analysis involved 89 nucleotide sequences. There were a total of 1501 positions in the final database. Bootstrap values are given at branches nodes and are based on 1000 replicates (values higher than 50% are indicated)

Full size image

In most isolates, the 16S rRNA gene sequence similarity is higher than 99.22% to a group of related species; only three isolates showed a value lower than 99.23% to known reference strains (Table 1). The names of the species closest to the isolates, percentages of similarity and accession numbers for the isolates are displayed in Table 1.

Table 1 Identification of chickpea seed endophytes by 16S rDNA sequencing and characterization of the plant growth-promoting activities
Full size table

Evaluation of Bacterial Endophytes’ Potential for Plant Growth Promotion

Isolates were evaluated for their plant growth promotion potential, namely tricalcium phosphate (TCP) and AlPO4 solubilization, siderophore, hydrogen cyanide (HCN) and indole-3-acetic acid (IAA) production (Table 1).

Siderophore production was the most widespread plant growth-promoting feature among the isolates (Fig. 2). For example, 25 isolates (71.4%) belonging to multiple genera, including Burkholderia, Mesorhizobium, Bacillus, Priestia, Paenibacillus, Tsukamurella, Microbacterium, Alcaligenes, and Enterobacter presented positive results for this plant growth-promoting characteristic (Fig. 2, Table 1). The Mesorhizobium sp. Y11C and Burkholderia sp. Y40C isolates showed greater siderophore producing ability (Table 1).

Fig. 2
figure 2

Percentage of isolates possessing (black bars) and lacking (gray bars) different plant growth-promoting traits, tricalcium phosphate (TCP) solubilization, aluminum phosphate (AlPO4) solubilization, siderophore, hydrogen cyanide (HCN) and Indole-3-acetic acid (IAA) production

Full size image

Regarding phosphate solubilization, 51.4% and 48.6% of the tested bacterial endophytes isolates showed positive results for TCP and AlPO4 solubilization, respectively (Fig. 2). Bacteria exhibiting phosphate solubilizing activity was observed in 40% of all isolates in both media (Table 1).

A high proportion (74.3%) of the endophytic bacterial isolates showed no ability to synthesize IAA-like molecules when grown in Yeast Extract Mannitol supplemented with 250 µg ml−1 of tryptophan (Fig. 2). The Alcaligenes sp. B4.3 isolate achieved the highest IAA-like molecules production (10.48 µg ml−1), followed by B. thuringiensis Y2B (9.35 µg ml−1), B. safensis Z12F (5.15 µg ml−1), and Paenibacillus sp. Y3B (2.02 µg ml−1) (Table 1). The remaining five isolates (Burkholderia sp. Z32C, Burkholderia sp. Y40C, B. safensis Z19C, Priestia megaterium Z20C, and Enterobacter ludwigii Z2F) produced less than 2 µg ml−1 of IAA-like molecules (Table 1).

Similar to IAA production, most of the isolates (94.3%) did not exhibit hydrogen cyanide (HCN) production (Fig. 2). Notably, only the B. thuringiensis Y2B and Acinetobacter sp. Z43C isolates displayed this feature (Table 1).

The majority (57.1%) of the endophytic bacteria possess two or more plant growth-promoting features, and 65% of them have three or more of the plant growth-promoting traits tested (Fig. 3). Among all isolates screened and characterized in this study, only B. thuringiensis Y2B exhibited the presence of all plant growth-promoting traits (Fig. 3, Table 1). In contrast, plant growth-promoting characteristics were considered absent in the B. safensis Z37F, B. safensis Z8F, B. safensis Y5F, and B. thuringiensis Z24C isolates (Table 1).

Fig. 3
figure 3

Five-ellipse Venn diagram presenting the number of isolates possessing each of the different plant growth-promoting traits, namely, phosphate solubilization, indole-3-acetic acid, hydrogen cyanide production, siderophore production

Full size image

Discussion

The microbial component of the plant holobiont has an important role in supporting plant growth and health [8]. Previous studies indicate that host species shape microbial community composition, plant structure and environmental factors; however, the extent to which each plays a role is unclear [16]. Highly complex microbial assemblages associated with specific plant tissues, such as roots, stems, leaves, fruits, tubers and seeds, were reported in different plant species [8]. Microbiota associated with the seed is acquired through horizontal and vertical transmissions, but the knowledge focused on the role and extension of seed microbiome on plant productivity remains largely unknown and underestimated [14, 15, 21, 30].

Seed borne bacteria with plant growth-promoting traits and biocontrol properties are in intimate contact with the plant tissues from an early stage and can contribute to the establishment and fitness of the host [14]. In this study, culturable endophytic bacteria were classified into the phyla Proteobacteria, Firmicutes, and Actinobacteria and identified as Mesorhizobium, Burkholderia, Alcaligenes, Acinetobacter, Rahnella. Enterobacter, Bacillus, Priestia, Paenibacillus, Tsukamurella, and Microbacterium. These phyla are possibly related to their predominance in soil and aquatic environments, suggesting that members of these phyla are more likely to be found during the plant life cycle [15]. Many of the bacterial genera identified herein were previously reported as seed endophytes in various plants. According to Johnston-Monje et al. [31], microbes inhabiting seed interiors and surfaces of 17 plant species were dominated by Proteobacteria and Ascomycetes, with the exceptions of coffee, soy, and Brachypodium, whose microbiomes were dominated by Firmicutes.

In legumes, studies on seed associated bacteria reported genera of Paenibacillus in pea [32], Bacillus, Staphylococcus, Enterococcus, Paenibacillus, Paracoccus, Brachybacterium, Acinetobacter, Micrococcus, and Kocuria in common bean [19], Agrobacterium, Aeromonas, Bacillus, Chryseomonas, Flavimonas, and Sphingomonas in soybean [33] and Staphylococcus, Pantoea, Mixta, Pseudomonas, and Bacillus in chickpea [10]. Interestingly, the last two taxa were also found in the present study.

Many of the bacterial genera encountered in this study have been reported as chickpea root endophytes with beneficial effects [11, 12, 34], and preliminary research on chickpea seed endophytes showed that Enterobacter was the dominant species [10]. In the present study, chickpea seeds had a significantly higher abundance of the phylum Firmicutes. Many species of this phylum are endospore-forming and possibly better adapted to withstand extreme environmental conditions and survive a long time in seeds [35]. Vertical transmission of Bacillus via seeds to the next generations was confirmed in other plants [36, 37].

A novel finding of this study is the presence of the Alphaproteobacteria Mesorhizobium sp. and the Betaproteobacteria Burkholderia sp. as endophytes of chickpea seeds. Studies on the diversity of rhizobia nodulating chickpea reported Mesorhizobium as the dominant microsymbiont in Europe, Africa, China and India, being Mesorhizobium ciceri, M. mediterraneum, M. amorphae, M. loti, M. huakuii, M. opportunistum, M. muleiense, M. tianshanense, M. wenxiniae, and M. plurifarium the main species identified [38]. Although preliminary, this study suggests that endophytic bacteria such as B. thuringiensis, Mesorhizobium sp., and Burkholderia sp. are vertically transferred to the next generation since analyzed seeds were originated from plants inoculated via seed coating with a mix of bacteria (Pseudomonas sp. DSM 33,393, Burkholderia sp. DSM 33,394, and Mesorhizobium sp. DSM 33,395) and sprayed with Bacillus thuringiensis subsp. aizawai at R5 and R7 growth stages. Previous studies that inoculated chickpea plants with the genera found in the present study reported improved plant growth, grain yield and grain protein content in previous studies [7, 12, 39, 40].

Revealing the composition, functionality and interaction of chickpea seed microbiota can lead to a better understanding of the role of the seed as a source of inoculum, which is important for practical applications to increase sustainable chickpea production. The present study provides evidence that culturable seed endophytes have a wide range of plant growth-promoting traits, including phosphate solubilization, siderophore, hydrogen cyanide (HCN) and indole-3-acetic acid (IAA) production that may benefit the host. Although P is abundant in soils, it is found in insoluble forms that are not accessible to plants, and it is one of the most limiting nutrients for crop productivity [41]. Several isolated strains converted insoluble forms of phosphorus to an accessible form, though the most remarkable results were shown by Acinetobacter sp. Z43C and Rahnella victoriana Z5F. Mukherjee et al. [10] reported that chickpea endophytes isolated from seeds could solubilize the insoluble phosphate and significantly increase plant growth. Siderophore production by endophytes is another important trait to improve plant nutrient uptake by increasing the bioavailability of Fe near the roots and consequently enhancing plant growth [42]. Moreover, microbial siderophores can trigger beneficial effects on plant defenses and act as an antagonistic mechanism by producing iron-chelating compounds under Fe-limiting conditions, thereby reducing the amount of iron available for the activity, colonization and proliferation of phytopathogens [43]. The high prevalence of siderophore production by Mesorhizobium species has been reported among chickpea microsymbionts [34]. In the present study, the isolate Mesorhizobium sp. Y11C showed the highest siderophores production level. Indole-3-acetic acid (IAA) is the most crucial natural and abundantly produced auxin, and it has been suggested that up to 80% of isolated rhizobacteria can synthesize this compound [44]. The prevalence of this trait was not observed among chickpea seed endophytes; however, B. safensis Z12F and Alcaligenes sp. B4.3 were able to produce more than 5 µg ml−1 of IAA-like molecules. Beneficial bacteria with this feature can stimulate root formation and development, enhancing plant drought stress tolerance [45]. Cyanide is a volatile secondary metabolite produced by rhizobacteria that may offer a selective advantage to the producer by out competing other microorganisms [46]. In the present study, only B. thuringiensis Y2B and Acinetobacter sp. Z43C produced HCN. Previous studies have demonstrated the suppression of chickpea root pathogens, such as Botrytis cinerea, Macrophomina phaseolina, Meloidogyne incognita, Fusarium oxysporum f. sp. ciceri, and Rhizoctonia solani by Pseudomonas spp. and Streptomyces spp. [47,48,49,50]. The authors speculated that plant growth stimulation and disease suppression are likely due to siderophore and HCN production or synergistic interactions between these two compounds or other metabolites produced by the microorganisms tested.

In this study, the majority of the chickpea isolates possess two or more plant growth-promoting mechanisms, with siderophore production and phosphate solubilization being the most common traits. Our observations are supported by previous studies that revealed chickpea microsymbionts, including seed endophytes, displaying plant growth-promoting traits with the ability to improve plant performance [10,11,12, 34]. In the present study, the isolate B. thuringiensis Y2B exhibited all plant growth-promoting traits evaluated and proved to be an ideal candidate for developing stable and efficient microbial inoculants for chickpea production. In previous studies, inoculation of chickpea with B. thuringiensis increased root dry weight and shoot dry weight by 30% and 18%, respectively, compared to non-inoculated plants [12]. Exploiting beneficial plant core microbiota is an emerging approach to increased sustainable production under normal and challenging conditions, significantly increasing the successful outcomes of biofertilizers and biopesticides.

Conclusion

This study revealed that chickpea seeds harbor highly diverse endophytic communities assigned to the phyla Proteobacteria, Actinobacteria, and Firmicutes. Bacillus was the dominant genus; however, rhizobia typically associated with chickpea roots were also found. Although preliminary, this study suggests evidence of vertically transmitted endophytes from seed to reproductive organs within the plants. The role of seed endophytic bacteria in shaping plant microbiome and transmission mode to the next generation through seeds is not completely clear. However, vertical transmission of bacterial species exhibiting plant growth-promoting traits fits well with the emerging view that recruitment, modulation and employment of the microbial component of the plant holobiont is a strategy to adapt agriculture production to a changing environment. Besides, most isolates possess multiple beneficial features that potentially contribute to nutrient acquisition and growth of the host, which opens new insights in the field of biofertilizers and high quality seeds production. Therefore, the development of microbial inoculants from plant core microbiota can increase the efficiency of biofertilizers and increase sustainable chickpea production, particularly in soils with low fertility or in areas where limited or restricted application of synthetic fertilizers are being imposed.