Impact of antagonistic endophytic bacteria on productivity of some economically important legumes

Abstract

Understanding the interactions within and between endophytes and their hosts is still obscure. Investigating endophytic bacterial plant growth–promoting (PGP) traits and co-inoculation effects on legumes’ performance is a candidate. Endophytic bacteria were isolated from Vicia sativa root nodules. Such endophytes were screened for their PGP traits, hydrolytic enzymes, and antifungal activities. Sterilized Vicia faba and Pisum sativum seedlings were co-inoculated separately with seven different endophytic bacterial combinations before being planted under sterilized conditions. Later on, several growth-related traits were measured. Eleven endophytes (six rhizobia, two non-rhizobia, and three actinomycetes) could be isolated, and all of them were indole-acetic-acid (IAA) producers, while seven isolates could solubilize phosphorus, whereas three, five, five, and four isolates could produce protease, cellulase, amylase, and chitinase, respectively. Besides, some of these isolates possessed powerful antifungal abilities against six soil-borne pathogenic fungi. Co-inoculation of tested plants with endophytic bacterial mixes (Rhizobia_mix+Actino_mix+non-Rhizobia_mix), (Rhizobia_mix+Actino_mix), or (Rhizobia_mix+non-Rhizobia_mix) significantly improved the studied growth parameters (shoot, root fresh and dry weights, length and yield traits) compared to controls, whereas co-inoculated plants with (Rhizobia_alone), (non-Rhizobia_mix), or (Actino_mix) significantly recorded lower growth parameters. Five efficient endophytes were identified: Rhizobium leguminosarum bv. Viciae, Rhizobium pusense, Brevibacterium frigoritolerans, Streptomyces variabilis, and Streptomyces tendae. Such results suggested that these isolates could be utilized as biocontrols and biofertilizers to improve legumes productivity. Also, co-inoculation with different endophytic mixes is better than single inoculation, a strategy that should be commercially exploited.

Supplementary Information

The online version contains supplementary material available at 10.1007/s42770-023-01204-x.

Introduction

For a long time, it was thought that leguminous root nodules were inhabited only by rhizobial bacteria. Later on, several microorganisms were separated from the majority of root nodules and were reported as nodule-associated microbiota [1]. Bacteria, actinomycetes, and fungi that reside internally in plant tissues are known as endophytic microbiomes [2]. Such internal plant-living microbes promote plant development and performance by mediating many essential metabolic interactions [3]. Endophytic bacteria showed a significant ability to reduce phytopathogens. The application of endophytic bacteria as biofertilizers is a practical option to enhance agricultural productivity, grain quality, and biodiversity of microbiota that has attracted a lot of interest in recent years [4].

Endophytic bacteria are believed to be beneficial to plant growth due to their plant growth–promoting (PGP) traits. Microbial indole-acetic-acid (IAA) performance, as an intermediate between microorganisms and plants, plays a role in the cross-talks between microbes and their host plants [5]. Also, phosphorus is very important for plant growth and plays a key role in many metabolic processes. Only 0.1% of soils’ phosphate could be used by plants, while the rest led to soil and groundwater pollution. Therefore, discovering microorganisms in the rhizosphere or within plants (endophytes) that could solubilize insoluble soil phosphate is a great challenge [6].

The use of microbes for disease control is of enormous value because it is eco-friendly, limits pathogens growth, improves soil quality, and preserves indigenous soil flora [7]. Endophytic bacteria could produce extracellular hydrolytic enzymes such as cellulase, xylanase, and amylase to resist pathogen invasion and acquire critical nutrients from their hosts [8].

The co-inoculation of leguminous plants with endophytic bacteria features important benefits such as a significant increase in nodulation, N₂ fixation, macro- and micro-element acquisition, plant growth, and productivity compared to using rhizobia alone [9].

The 16S ribosomal RNA (16S rRNA) gene sequence is generally used for the identification and classification of microbes. Also, it affords enough phylogenetic information and relationships to identify the isolates down to the genus level, using the large 16S rRNA gene sequence database information that is publicly reachable [10].

This study focused on discovering the features, importance, and efficacy of the endophytic bacteria that were isolated from Vicia sativa root nodules. Moreover, how did these endophytes influence the performance of the two tested legumes, viz., Vicia faba and Pisum sativum, when co-inoculated in different regimes? Moreover, the five most efficient isolates were identified at the species level.

Materials and methods

Isolation and characterization of endophytes’ PGP traits

V. sativa L. (Vetch) seeds and plants were collected in March 2020 at the flowering and/or fruiting stage from El-Alamein Italian cemeteries, 5 km west of El-Alamein city (30° 50′ 0″ N, 28° 57′ 0″ E). Endophytic bacteria (rhizobia (R), non-rhizobia (non-R), and actinomycetes (Ac)) were isolated and purified from root nodules as described before [11] undamaged nodules were surface-sterilized being immersed for 5–10 s in 95% ethanol, subsequently immersed for 4–6 min in a 3% hydrogen peroxide solution, and finally washed repeatedly with sterile water. In a sterile test tube containing 1 ml of sterile distilled water, a sterile steel ball was used to shatter the surface-sterilized nodule. On YEMA plates, the single loop of nodule drop was scattered containing Congo red (CR).

Plates were scattered and incubated for 3–10 days at 28–30 °C until well-isolated colonies appeared. Each observed isolate was selected and re-streaked for more purification.

Indole-acetic-acid (IAA) production was measured calorimetrically at 530 nm (micrograms per milliliter) [12]. Endophytes were screened for their capacity to solubilize dibasic calcium phosphate (CaHPO₄) using Pikovskaya’s phosphate medium (PVK) [13], the National Botanical Research Institute’s phosphate growth medium (MNBRI) [14], or yeast extract mannitol (YEM) [15]. After 1 week, those that caused phosphate solubilization in the clear zone were considered P-solubilizing isolates.

The isolated endophytes were tested for their capability to synthesize hydrolytic enzymes such as protease, cellulase, amylase, and chitinase, as described by [16]. The formation of a distinct halo zone surrounding the developed colony was regarded as a positive signal for enzyme production.

Also, the antifungal activities of such endophytes were screened as described by [17] against eight soil-borne pathogenic fungi, viz., Sclerotinia sclerotiorum (11690), Fusarium circinatum (11691), Fusarium subglutinans (11692), Alternaria alternata (11694), Aspergillus brasiliensis (11695), Rhizopus stolonifer (11696), Nigrospora oryzae (13340), and Scopulariopsis brevicaulis (13341). These tested fungi were identified at the Mycological Center at Assiut University (AUMC). Across fungal and endophytic bacterial colonies, the inhibitory clear zones were measured.

Co-inoculation of some legumes with endophytes

Greenhouse experiments were conducted during the period of December 2020 to January 2021 (24/15 °C, 11/13 h day/night regimes) in a randomized block design where the block size was 60 cm L × 50 cm W in 3 replicates. V. faba (broad bean) and P. sativum (pea) seeds were scarified (the first one only) and sterilized before germination as described by [18]. After germination on sterilized 1% (w/v) water agar, the seedlings were transplanted into sealed plastic pots containing autoclaved sterilized clay sandy soil (1:1 v/v). For the co-inoculation survey, the seedlings were injected independently with 1 ml per seedling using one of the following treatments in Table 1.

Table 1.

Different treatments of the endophytic rhizobial and non-rhizobial bacteria, where R_mix = a mixture of six different rhizobial isolates; non-R_mix = a mixture of two non-rhizobial; and AC_mix = a mixture of three different actinomycete isolates

Number	Co-inoculation of seeds (treatments)
1	Ralone (40.1R)
2	(Rmix), (ACmix), or (Non-Rmix)
3	(Rmix+ACmix+Non-Rmix), (Rmix+ACmix), or (Rmix+Non-Rmix)
4	Seeds without inoculation (as a control)

Endophytic bacterial isolates were incubated in YEM medium until the late growth stage before inoculation. Both treated and untreated seeds of the two tested plant species (V. faba and P. sativum) were grown to maturity. Plants were irrigated with a 50% diluted sterilized Jensen solution weekly in exchange for water [11].

A number of growth-related traits (ten replicates, one treatment, and one host) were measured after 50 days of planting. The shoot-related traits included shoot fresh weight (ShFWt), shoot dry weight (ShDWt), water content (WC), and shoot length (ShL), while the root-related traits included root fresh weight (RoFWt), root dry weight (RoDWt), water content (WC), and root length (RoL). DWt was obtained by drying the fresh samples at 105 °C for 48 h to a constant weight. The WC of the plants was calculated using the formula [(FW-DW)/FW] × 100. At maturity, some yield components were measured, including pods’ fresh weight/plant (PsFWt/Pl), seeds’ fresh weight/plant (SFWt/Pl), and seeds’ number/plant (SNo/Pl) [19]. The data were analyzed statistically by ANOVA using IBM SPSS Statistics 23. The test of significance was carried out using the TUKEY test at the significance level of P ≤ 0.05.

Molecular characterization of the endophytic bacterial isolates

DNA was extracted from 20 ml of YEM endophytic bacterial culture after 7 days of incubation [20]. PCR amplification of the 16S rRNA gene was done using the primer sequences fDl and rD1, as described previously [21].

PCR products were further purified with the QIAquick PCR purification kit (Qiagen), sequenced directly using the same primers as for PCR amplification. The sequence was generated in an ABI 3730XL DNA Analyzer-sequencer (Applied Biosystems), with insertion and electrophoresis parameters of 1 Kv/90 s and 7 Kv/240 min, correspondingly to [22] at Solgent Co Ltd (South Korea). The sequences obtained were compared with those from GenBank using the BLASTN program [23] to identify the isolates. The phylogenetic analysis was performed with the MEGA X program (version 10.1.8) for isolates’ 16S rRNA gene sequences [22] using the neighbor-joining algorithm. The data were submitted to the NCBI GenBank database to be publicly available and to obtain an accession number.

The NCBI GenBank accession numbers for the 16S rRNA of strains Rhizobium leguminosarum bv. Viciae, Agrobacterium pusense (Rhizobium pusense), Peribacillus frigoritolerans (Brevibacterium frigoritolerans), Streptomyces variabilis, and Streptomyces tendae are MT917183.1, MT917196.1, MT917022.1, MT918391.1, and MT918401.1, respectively.

Infectivity test

Seeds of the two investigated leguminous plants (V. faba and P. sativum) were sterilized, germinated, transplanted, co-inoculated, and treated as explained previously [11]. These plants were screened for nodule formation after 7 weeks.

Results

Isolation and characterization of endophytes PGP traits

Six rhizobial (R), two non-rhizobial (non-R), and three actinomycete (AC) bacterial isolates were obtained from root nodules of V. sativa (L.).

IAA production was detected from all the isolates, with different intensities ranging from 1.84 to 1.94 μg ml⁻¹ from 6.3*10⁷ cfu ml⁻¹ to 8.5*10⁵ cfu ml⁻¹ for isolates 40.6non-R and 70.1R, respectively (Supplementary Table 1). Only seven isolates [20.1R, 30.1R, 40.1R, 50.1R, 70.2R, 40.5non-R, and 40.4AC] were able to solubilize P on MNBRI media, producing 0.9-, 0.8-, 0.8-, 0.6-, 0.8-, 0.4-, and 0.9-cm clear zones, respectively (Supplementary Table 1), while all isolates failed to solubilize dibasic calcium phosphate (CaHPO₄) on PVK or YEM media.

The 11 endophytic bacterial isolates were screened for lytic enzyme production and antibiosis activity. For protease production, three endophytic isolates were positive, giving halo zones of 0.5, 0.8, and 0.9 cm for isolates 40.5non-R, 40.2AC, and 40.3AC, respectively (Supplementary Table 1). For cellulase production, five endophytic isolates named 20.1R, 30.1R, 40.1R, 40.5non-R, and 40.6non-R were positive, causing a 0.1-cm small halo zone. For amylase production, five endophytic isolates, viz., 40.5non-R, 40.6non-R, 40.2AC, 40.3AC, and 40.4AC, were positive giving clear zones of 0.1, 0.1, 1.1, 0.5, and 0.4 cm, respectively. For chitinase production, four endophytic isolates, viz., 40.5non-R, 40.2AC, 40.3AC, and 40.4AC, were positive, giving clear halo zones of 0.1, 0.2, 0.2, and 0.4 cm, respectively (Supplementary Table 1).

Regarding the antifungal activity, five isolates, viz., 70.1R, 40.6non-R, 40.2AC, 40.3AC, and 40.4AC, were able to antagonize S. sclerotiorum (Fig. 1 A1-4), with 3.3-, 3.1-, 4.2-, 3.9-, and 4.2-cm inhibition zones, respectively (Supplementary Table 1). On the other hand, nine positive endophytic bacterial isolates, viz., 20.1R, 30.1R, 40.1R, 50.1R, 70.1R, 70.2R, 40.2AC, 40.3AC, and 40.4AC, were able to antagonize F. circinatum (Fig. 1 A5-8), F. subglutinans (Fig. 1 A9-12), and A. alternate (Fig. 1 A13-16) with different inhibition zones ranging between 0.3 and 3.2 cm (Supplementary Table 1). For N. oryzae, only four isolates, viz., 70.1R, 40.2AC, 40.3AC, and 40.4AC, showed an antagonistic effect (Fig. 1 A17-20) with 2.6-, 2.9-, 2.6-, and 2.8-cm inhibition zones, respectively. All the endophytic isolates could antagonize S. brevicaulis (Fig. 1 A21-24) with inhibition zones varying from 2.1 to 4.0 cm for isolates 40.6non-R and 40.2AC, respectively. Finally, all the endophytic isolates failed to antagonize A. brasiliensis or R. stolonifer in vitro.

Fig. 1.

Antifungal abilities of the endophytic bacterial isolates: 70.1R (3, 7, 11, 15, 19 & 23); 40.2AC (2, 6, 10, 14 & 18); 40.3AC (5, 9, 13, 17 & 21) and 40.4Ac (1 & 22) against six soil-borne pathogenic fungi: S. scleotiorum (1-4); F. circinatum (5-8); F. subglutinans (9-12); A. alternata (13-16); N. oryzae (17-20) and S. brevicaulis (21-24). Cont = fungal growth as control. R = rhizobia and AC = actinomycetes

Co-inoculation and plant growth performance

The growth performance of the two tested plant species, V. faba and P. sativum, when co-inoculated with rhizobial isolates or non-rhizobial and/or actinomycete isolates recorded magnificent results. Co-inoculation of plants with (R_mix+Ac_mix+non-R_mix), (R_mix+Ac_mix), and (R_mix+non-R_mix) significantly increased ShFWt of 9±2.2 g, 8±1.2 g, and 7±2.2 g for V. faba and 6.9±1.5 g, 9±2.2 g, and 6.6±1.3 g for P. sativum, compared to control plants of 1.6±0.9 g and 1.6±0.9 g, respectively. On the other hand, (R_alone)-, (AC_mix)-, and (non-R_mix)-co-inoculated plants significantly recorded lower ShFWt of 2±0.8 g, 4±1.2 g, and 3±1.2 g for V. faba and 2±1 g, 4±1.6 g, and 3±1.2 g for P. sativum, respectively, compared to plants co-inoculated with endophytic mixes (Fig. 2).

Fig. 2.

Co-inoculation effects of endophytes on shoot fresh weight (ShFWt) of Vicia faba (V.f.) and Pisum sativum (P.s.). Bars followed by the same lowercase letters are not significantly different at 0.1E⁻¹² < P < 0.05 by UNIANOVA

In terms of RoFWt, plants co-inoculated with (R_mix+AC_mix+non-R_mix), (R_mix+AC_mix), and (R_mix+ non-R_mix) significantly recorded the highest RoFWt of 9±1.8 g, 10±1.6 g, and 8±2.3 g for V. faba and 2.1±1 g, 2.9±1.4 g, and 1.7±1.1 g for P. sativum, compared to control plants of 2±1.1 g and 0.3±0.3 g, respectively. Nevertheless, plants co-inoculated with (R_alone), (AC_mix), and (non-R_mix) were significantly lower than plants co-inoculated with endophytic mixes to 2±1 g, 5±2.1 g, and 3±1.3 g for V. faba and 0.5±0.3 g, 1±0.7 g, and 0.8±0.2 g for P. sativum, respectively (Fig. 3). The performance of ShDWt, RoDWt, and WC responding to the different co-inoculation regimes was more or less in the same pattern as the fresh weight data (data not shown).

Fig. 3.

Co-inoculation effects of endophytes on root fresh weight (RoFWt) of Vicia faba (V.f.) and Pisum sativum (P.s.). Bars followed by the same lowercase letters are not significantly different at 0.1E-17 < P < 0.05 by UNIANOVA

RoL was significantly higher with treatments (R_mix+AC_mix+non-R_mix), (R_mix+AC_mix), and (R_mix+non-R_mix) of 25±1.2 g, 27±1.6 g, and 23±1.2 g for V. faba and 12±2.2 g, 15±2.5 g, and 11±1.5 g for P. sativum, compared to control plants of 11.3±2.2 g and 4±1.7 g, respectively. No significant differences were recorded among the three treatments R_alone, R_mix, and non-R_mix (Fig. 4).

Fig. 4.

Co-inoculation effects of endophytes on root length (RoL) and shoot length (ShL) of Vicia faba (V.f.) and Pisum sativum (P.s.). Bars followed by the same lowercase letters are not significantly different at 0.1E^-5 < P < 0.05 by UNIANOVA

Regarding ShL, plants co-inoculated with (R_mix+AC_mix+ non-R_mix)_, (R_mix+AC_mix), and (R_mix+non-R_mix) significantly exceeded the other treatments, being 16.9±1.5 g, 17.2±0.7 g, and 15±0.9 g for V. faba and 5±2.2 g, 7±1.4 g, and 6±1.2 g for P. sativum, respectively. ShL for plants co-inoculated with (R_alone), (AC_mix), or (non-R_mix) were significantly lower, being 9.5±1.3 g, 10±1.4 g, and 11±2.2 g for V. faba or 2±0.8 g, 4.2±2.5 g, and 3.1±2 g for P. sativum compared to plants co-inoculated with endophytic mixes (Fig. 4).

Concerning yield traits, for V. faba plants, PsFWt/Pl, SFWt/Pl, and SNo/Pl significantly increased when co-inoculated with (R_mix+AC_mix+non-R_mix), (R_mix+AC_mix), and (R_mix+non-R_mix), recording (6.38±2.0 g, 5.5±1.0 g, and 4.38±1.5g), (2.6±0.8 g, 2±1.1 g, and 2.14±1.0 g), and (5±1.7, 3±1.7, and 4±1.4), respectively (Table 2) compared to controls (1.8±0.7 g, 0.78±0.1 g, and 1±0.0). Co-inoculation of P. sativum plants with (R_mix+AC_mix+non-R_mix), (R_mix+AC_mix), and (R_mix+non-R_mix) significantly increased PsFWt/Pl, SFWt/Pl, and SNo/Pl, being (2.9±0.8 g, 2.5±0.5 g, and 2±0.0 g), (1.28±0.2 g, 1.5±0.4 g, and 1.25±0.2 g), and (4±0.9, 6±1.3, and 3±0.6), respectively (Table 2) compared to their controls (0.6±0.1 g, 0.2±0.0 g, and 1±0.0). The other treatments, viz., R_alone, R_mix, AC_mix, and non-R_mix, could not significantly improve the majority of yield traits either compared to endophytic mixes or their controls in the two tested plants (Table 2).

Table 2.

Co-inoculation effects of endophytes on pods’ fresh weight/plant (PsFWt/Pl), seeds’ fresh weight/plant (SFWt/Pl), and seeds’ number/plant (SNo/Pl) of Vicia faba (V.f.) and Pisum sativum (P.s.). Results are the mean in grams or number (No.) ± SD. Numbers followed by the same lowercase letters are not significantly different at 0.1E⁻⁷ < P < 0.05 by UNIANOVA

Treatments	V.f.			P.s.
	PsFWt/Pl	SFWt/Pl	SNo/Pl	PsFWt/Pl	SFWt/Pl	SNo/Pl
Ralone	2.00±2.0c	0.94±0.1bc	2±1.1bc	1.00±0.0c	0.40±0.1d	2±0.6c
Rmix	3.19±0.9bc	1.10±0.5bc	2±0.6bc	1.79±0.9b	0.74±0.2c	2±0.6c
Rmix+ACmix+ non-Rmix	6.38±2.0a	2.60±0.8a	5±1.7a	2.90±0.8a	1.28±0.2a	4±0.9b
Rmix + ACmix	5.50±1.0ab	2.00±1.1ab	3±1.7abc	2.50±0.5ab	1.50±0.4a	6±1.3a
Rmix + non-Rmix	4.38±1.5ab	2.14±1.0ab	4±1.4ab	2.00±0.0ab	1.25±0.2ab	3±0.6bc
ACmix	3.20±1.4bc	1.21±0.4bc	2±1.1bc	1.50±0.3bc	0.90±0.1bc	3±0.9bc
Non-Rmix	3.00±1.7bc	1.26±0.4bc	3±1.7abc	1.70±0.6b	0.80±0.1c	4±0.9b
Control	1.80±0.7c	0.78±0.1c	1±0.0c	0.60±0.1c	0.20±0.0d	1±0.0d

Molecular characterization of endophytic bacterial isolates

The 16S rRNA gene of the five efficiently selected endophytic bacterial isolates proved to be the expected PCR product at 1500 bp. The nearly complete sequences of the 16S rRNA gene showed the closest relative species to be Rhizobium leguminosarum bv. Viciae (MT917183.1), Rhizobium pusense (MT917196.1), Brevibacterium frigoritolerans (MT917022.1), Streptomyces variabilis (MT918391.1), and Streptomyces tendae (MT918401.1), corresponding to the five efficiently selected endophytic bacterial isolates of V. sativa L. (40.1R, 70.1R, 40.5non-R, 40.3AC, and 40.4AC), respectively. A phylogenetic tree developed from 16S rRNA gene sequences using the MEGA X indicated that three strains, including Brevibacterium frigoritolerans (MT917022.1), Streptomyces variabilis (MT918391.1), and Streptomyces tendae (MT918401.1) belonging to the phylum Actinobacteria, were closely clustered together with the type strains CP030063.1, NR 043840.1, and KY072953.1, respectively (Fig. 5), while the other two strains, Rhizobium leguminosarum bv. Viciae (MT917183.1) and Rhizobium pusense (MT917196.1) belonging to phylum the Proteobacteria, were grouped with type strains CP022665.1 and CP053857.1 (Fig. 5).

Fig. 5.

Neighbor-joining phylogenetic tree based on partial 16S rRNA gene sequences of the five selected endophytic bacterial strains (within rectangles) in comparison to reference sequences from NCBI. The scale bar indicates 0.03% nucleotide substitutions. The accession numbers are listed after the species name. Next to the branches are the percentage of duplicate trees in which the related taxa clustered together in the bootstrap test (1000 repetitions)

Infectivity test

Co-inoculation with (R_alone), (R_mix), (R_mix+AC_mix+Non-R_mix), (R_mix+AC_mix), or (R_mix+Non-R_mix) produced effective symbiosis with V. faba and P. sativum after being isolated from the root nodules of V. sativa (the main host). The other treatments (AC_mix) or (Non-R_mix) failed to form any nodules with V. faba or P. sativum. All the previously mentioned legumes did not show any nodules when planted in the autoclaved, sterilized, and not inoculated soils.

Discussion

V. sativa, grown wildly, was selected to investigate the relations within and between endophytes inhabiting its nodules and some economical legumes as well. The efficacy of these isolates was studied by recording several endophytic PGP and growth-related traits of the tested legumes when co-inoculated with endophytes in different combinations. From V. sativa root nodules, rhizobial bacteria, non-rhizobial bacteria, and actinomycetes were isolated and recognized as endophytes. Such endophytic diversity was previously recorded [24], where rhizobial and non-rhizobial endophytes were isolated from root nodules of Lupinus angustifolius growing in Northern Tunisia.

Agricultural microbiologists were interested in finding efficient endophytic bacteria able to improve IAA production and phosphorous availability for better plant growth and development [25]. Our investigations showed that all the isolates were IAA producers, while seven out of the eleven tested endophytes could solubilize CaHPO₄.

In this study, 27%, 45.5%, 45.5%, and 36.4% of the screened endophytic bacterial isolates were positive for protease, cellulose, amylase, and chitinase production, respectively, while 15.5%, 9.5%, 4%, and 3.5% of the tested isolates produced these hydrolytic enzymes [16]. The role and importance of such hydrolytic enzymes arise from their ability to prevent pathogen growth, thus decreasing crop damage [26].

In the current study, 45.5% of isolates were able to antagonize S. scleotiorum. While 81.8% of isolates were able to antagonize F. circinatum, F. subglutinans, and A. alternata. Also, 36.4% of isolates were capable of antagonizing N. oryzae. Moreover, all the endophytic isolates could antagonize S. brevicaulis, whereas all endophytic isolates could not antagonize A. brasiliensis and R. stolonifer in vitro. Previously, more than 70% of isolates inhibited the growth of both Pythium aphanidermatum and Rhizoctonia solani [27]. The importance of finding such antifungal endophytic bacteria arises from their role in eliminating specific plant-harmful fungi by causing significant alterations and mycelial destruction [27, 28].

In this study, plant growth–related traits improved significantly when the investigated plants (V. faba and P. sativum) were co-inoculated with (R_mix+AC_mix+ non-R_mix) or (R_mix+AC_mix) or (R_mix+ non-R_mix) rather than single inoculations (R_alone, AC_mix or non-R_mix) or without inoculations (control plants). Such results were supported previously, where shoot and root dry biomass increased significantly when plants were co-inoculated with Streptomyces griseoflavus and Bradyrhizobium elkanii [29] or Azospirillum brasilense and Bradyrhizobium strain 15A [30]. Also, legumes’ co-inoculation with non-rhizobial bacteria has synergistic effects on plant growth and yield that are different from those of single inoculation [31]. The advantages of using effective inoculants as biofertilizers in agriculture are an alternative approach to minimizing the hazards of synthetic fertilizers [32].

The most significant endophytic bacterial isolates (in this study) were identified as Rhizobium leguminosarum bv. Viciae (40.1R) and Rhizobium pusense (70.1R), which belong to the phylum Proteobacteria. In addition, three endophytic bacterial isolates were identified: Brevibacterium frigoritolerans (40.5non-R), Streptomyces variabilis (40.3AC), and Streptomyces tendae (40.4AC) that belong to the phylum Actinobacteria. Similarly, the 16S rRNA gene sequence was used to identify 18 selected out of 40 pigeon peas isolates [33]. Also, previous studies on nodules of native legumes showed that not are only they occupied by highly diverse rhizobia but also by other bacteria [34].

Conclusions

The isolated strains could be used as bio-inoculants to raise the resistance of plants to biotic and abiotic stresses, to improve legumes productivity, and as biofertilizers. Also, this study recommends the application of different combinations (mixes) of Rhizobium spp., non-rhizobial bacteria, and actinomycetes species as a strategy that should be commercially exploited. Such a strategy will provide legume plants with a complete benefits package.

Declarations

Competing interests

The author declare no competing interests.