Soybean [ Glycine max (L.) Merr.], is one of the most cultivated legume worldwide due to its high protein content and important industrial by-products ( Stacey et al. , 2004 ; Masuda & Goldsmith, 2009 ). Nitrogen is one of the macro-nutrients required for growth and development of plants, so this nutrient must be supplied via chemical fertilization and/or biological N fixation (BNF) ( de Carvalho et al. , 2013 ; Gai et al. , 2017 ). For soybean, the BNF is carried out in root nodules by symbiotic bacteria known as rhizobia. Rhizobia obtain carbon sources from plants and in return, the bacte­rial nitrogenase enzyme activity provides ammonia to plants ( Santi et al. , 2013 ). The symbiosis between soybean and rhizobia is a complex process involving activities of several genes ( de Carvalho et al. , 2013 ; Lira et al. , 2015 ). Many legumes form root nodules that may stimulate plant growth when nodules are formed by compatible and functional rhizobial strains. In this regard, the bacterial establishment is highly specific, but strongly affected by genetic bacterial/plant interactions that determine an efficient symbio­tic relationship ( Boonkerd & Singleton, 2002 ). This symbiosis is also largely dependent on environmental conditions ( Valencia et al. , 2010 ).

Most soybean varieties fail to nodulate efficiently in tropical soils even though plants are inoculated with competitive rhizobial strains ( Kueneman et al. , 1984 ). In this sense, native rhizobia most likely promote greater plant growth since they are more adapted to environmental conditions ( Waluyo et al. , 2005 ; Soe & Yamakawa, 2013 ). Thus, the isolation and re-introduction of highly competitive and effective native rhizobia are important for increasing soybean production.

Besides rhizobia, root nodules may host other endophytic non-symbiotic bacteria ( Li et al. , 2008 ; Saïdi et al. , 2011 ; Aserse et al. , 2013 ), and these bacteria are unable to form nodules nor perform N-fixation. However, these endophytic non-symbiotic bacteria may favor plant growth and nutrition, or assist the solubilization of insoluble forms of phosphates in the rhizosphere ( Bai et al. , 2002 ; Li et al. , 2008 ; Liu et al. , 2010 ; Stajkovic et al. , 2011 ; Aserse et al. , 2013 ). Co-inoculation of endophytic non-symbiotic bacteria along rhizobia has gained special interest as part of sustainable agriculture, since both bacteria may act synergistically for enhancing legume growth and performance, in comparison to the single inoculation of rhizobia ( Bai et al. , 2002 ). For instance, Rhizobium phaseoli co-inoculated with either Pseudomonas sp. or Bacillus sp. Bx, resulted in significant increase in the stem dry weight of beans ( Stajkovic et al. , 2011 ). Similarly, the co-inoculation of Mesorhizobium gobiense with Bacillus pumilus B402 resulted in increased number of nodules and growth of Sphaerophysa salsula (Pall.) DC. ( Deng et al. , 2011 ). Also, an increased root nodulation in Medicago sativa L. was reported due to the co-inoculation of Sinorhizobium meliloti and Agrobacterium tumefaciens ( Wang et al. , 2006 ). Similarly, an increase in nodulation of Wisteria sinensis (Sims) DC. was observed by combining S. meliloti and Agrobacterium sp. II CCBAU 21244 ( Liu et al. , 2010 ). In the case of soybeans, the co-inoculation of Bradyrhizobium japonicum with Bacillus subtilis or B. thuringiensis increased both plant weight and nodulation ( Bai et al. , 2002 , 2003 ). In contrast, Camacho et al. (2001) reported decreases in root nodulation of soybean due to the co-inoculation of B. japonicum USDA110 and Bacillus sp. CECT450. The contrasting effects of bacterial co-inoculation on soybean indicate the necessity for identifying efficient combinations of rhizobia and other endophytic strains to promote plant growth and yield, thus, reducing the application of high doses of chemical fertilizers. So, it is important to explore endophytic bacterial strains cohabiting soybean nodules, as they may contribute to growth promotion in legumes. Consequently, the latter allows the reduction of environmental pollution and promotes sustainable agriculture of soybean in tropical regions.

The objectives of this work were to: 1) isolate and characterize symbiotic and endophytic non-symbiotic bacteria from root nodules of Glycine max collected from two tropical cropping systems at Campeche, Mexico, and 2) evaluate the effects of the most prominent endophytic bacterial strains when co-inoculated with referential or native rhizobial strains on the growth of soybean plants. The results are expected to contribute on selecting an efficient combination of rhizobia and endophytic bacteria for being used as biofertilizers for soybean cultivation in tropical conditions.

The legume-rhizobia symbiosis is an important biological process for plant productivity ( Bai et al. , 2003 ; Stajkovic et al. , 2011 ). Many studies have shown that the co-inoculation of rhizobia and some endophytic bacteria may contribute on plant growth promotion and yield ( Bai et al. , 2003 ; Liu et al. , 2010 ; Deng et al. , 2011 ); thus, such co-inoculation may improve the effectiveness of the symbiotic relationship. In this context, the nodules are colonized by several non-rhizobial endophytes ( Bai et al. , 2002 ; Palaniappan et al. , 2010 ; Saïdi et al. , 2011 ; Li et al. , 2012 ; Aserse et al. , 2013 ) which influence the growth and yield of legumes by different mechanisms such as mineral solubilization, or enhanced root nodulation and N fixation activity ( Bai et al. , 2003 ; Palaniappan et al. , 2010 ; Deng et al. , 2011 ; Stajkovic et al. , 2011 ; Li et al. , 2012 ). In the present study, 23 strains (predominantly Gram-negative bacteria) were isolated. Of them, 19 were found as non-symbiotic endophytes, and 4 showed symbiotic features. In this respect, Li et al. (2008) isolated a high number of Gram-negative endophytes from soybean nodules. In contrast, other studies reported low number of endophytic bacteria with the predominance of Gram-positive bacteria ( Bai et al. , 2002 ; Hung & Annapurna, 2004 ; Aserse et al. , 2013 ). Therefore, our study besides isolating three genera of potentially nodule-forming rhizobia, also reports the proliferation of 7 genera of non-symbiotic bacteria harbored in root nodules.

The analysis of the 16S rRNA gene sequence of the isolated strains (symbiotic and non-symbiotic) indicated that they belonged to 10 different genera (Table 2). Among the isolated bacteria, Agrobacterium tumefaciens is known as a soil-borne phytopathogen previously reported from soybean nodules ( Li et al. , 2008 ). Same authors also reported the genus Serratia in the soybean nodules, and this bacterium has been shown to stimulate the growth and development of soybean ( Zhang et al. , 1996 ); however, the two Serratia strains isolated in this work did not show significant effects on growth nor on soybean nodulation. Our report revealed that bacterial genera contrast with other findings in which other genera of endophytic bacteria, such as Acinetobacter, Bacillus, Burkholderia, Deinococcus, Rhodococcus, Pantoea, Staphylococcus and Tsukamurella , were found in soybean nodules ( Bai et al. , 2002 , 2003 ; Hung & Annapurna, 2004 ; Li et al. , 2008 ; Aserse et al. , 2013 ).

Our study also noted the isolation of other endophytic bacteria not previously described as inhabitants of soybean nodules, such as Enterobacter, Chryseobacterium, Massilia, Microbacterium , and Xanthomonas . However, these bacteria were reported as nodule inhabitants of other legume species; for example, Enterobacter was identified from nodules of Abrus precatorius and Vigna unquiculata ( Ghosh et al. , 2015 ; Leite et al. , 2016 ), Chryseobacterium from V. unquiculata ( Leite et al. , 2016 ), Massilia from nodules of Hedysarum flexuosum ( Ezzakkioui et al. , 2015 ), Microbacterium from Medicago sativa and Sphaerophysa salsula ( Stajkovic et al. , 2009 ; Deng et al. , 2011 ), Serratia from nodules of Sphaerophysa salsula and Hedysarum flexuosum ( Deng et al. , 2011 ; Ezzakkioui et al. , 2015 ), and Xanthomonas was iden­tified in nodules of Medicago hispida ( Arone et al. , 2014 ). More importantly, the influence of these bacteria on the symbiosis between rhizobia and soybean has been rarely described.

In the present study, the co-inoculation of non-symbiotic endophytic bacteria did not produce significant effects on the growth of soybean plants. In contrast, both dry weight and root nodulation were increased due to the co-inoculation of B. japonicum 532C with the endophytic Bacillus subtilis and B. thuringien­sis ( Bai et al. , 2002 ). Similarly, the coinoculation of S. meliloti with endophytic bacteria like A. tumefaciens ( Wang et al. , 2006 ) or Rhizobium sp. II CCBAU21244 ( Liu et al. , 2010 ) resulted in increased nodulation of Melilotus dentatus and Wisteria sinensis . Nevertheless, in our work, the co-inoculation of A. tumefaciens CPO4.15C did not affect root nodulation which is opposite to results obtained by Camacho et al. (2001) . Overall, the endophytic bacteria isolated in the present study did not influence plant growth, however, they may be involved in creating an ecological micro-niche suitable for both survival and proliferation of symbiotic bacteria, as discussed by Deng et al. (2011) .

On the other hand, the symbiotic strain Brady­rhizobium sp. CPO4.24C was able to form nodules in the soybean plants. On the contrary, the absence of nodules in the 'Huasteca 200' variety inoculated with Rhizobium sp. CPO4.13C and Ensifer adherensis CPO4.2TA or CPO4.18T can be explained due to the specificity between legumes and rhizobia; in this regard, these two bacterial genera are not soybean symbionts ( Wu et al. , 2011 ; Zhang et al. , 2011 ; Yan et al. , 2014 ). Similarly, some non-nodulating Rhizobium and Bradyrhizobium bacteria were reported in the rhizosphere of legumes ( Segovia et al. , 1991 ; Pongsilp et al. , 2002 ; Aserse et al. , 2013 ).

As mentioned, the inoculation of Bradyrhizobium sp. CPO4.24C resulted in greater nodulation in comparison to the inoculation of the reference strain B. japonicum USDA110 (Table 3). This reference strain has induced abundant nodulation at low temperatures ranging between 17 to 23ºC ( Ando & Yokoyama, 1999 ; Suzuki et al. , 2014 ). Nevertheless, the average temperature recorded in the present study was 27 ± 2ºC by which the growth and infectivity of the reference bacterial strain might have been affected. However, the co-inoculation of both strains resulted in high PH and nitrogenase activity (consequently greater N fixation can be expected) when compared to the single inoculation of each strain. This demonstrates a synergistic effect produced by both of the aforementioned bacteria. In this respect, Htwe & Yamakawa (2016) reported lower ARA (1.15 C ₂ H ₄ h ^-1 plant ^-1 ) due to the co-inoculation of soybean plants with B. japonicum SAY3-7 and Streptomyces griseoflavus P4. In addition, these authors reported that SDW and RDW were significantly higher in co-inoculated plants (0.42 and 0.25 g/plant) than uninoculated controls (0.39 and 0.25 g/plant). Similarly, in this study the shoot and root biomass were enhanced with the co-inoculation of Bradyrhizobium sp. CPO4.24C and B. japonicum USDA110 in comparison to uninoculated control (Table 4).

Our results show that the native strain Brady­rhizobium sp. CPO4.24C has good potential for being introduced as biofertilizer for soybean cultivation in the tropical regions of Mexico. Nevertheless, further research should be conducted for evaluating the effects of this bacterium on the growth and yields of soybean plants under appropriate field conditions.

Overall, this study isolated twenty-three endophytic bacterial strains belonging to ten different genera from nodules of four varieties of Glycine max grown at field conditions. Furthermore, co-inoculation of the three prominent bacterial endophytes with either native or referential Bradyrhizobium strains did not enhance plant growth nor root nodulation. The native Bradyrhizobium sp. CPO4.24C showed high potential for being inoculated alone or combined with the referential strain B. japonicum USDA110, since PH, nodulation, dry weight, relative chlorophyll content, and nitrogenase activity were significantly enhanced.