Among the plant growth promoting hormones produced by microorganisms, indole acetic acid is the most common and physiologically active27. The results from the present study showed that the tested bacterial strains were able to synthesize IAA in vitro. Six of the bacterial isolates under study exhibited their positive reaction by developing pink colour when reacted with Salkowski’s reagent which indicates positive result for IAA production. Further, the results indicated that the microbes differed in their ability to produce IAA. Earlier studies have shown that IAA production by microbes differed between different species or even within strains of the same species28,29,30. From the ten bacterial species and strains tested in this study, the Rhizobium produced higher levels of IAA compared to the Pseudomonas and Paenibaccillus species. This concurs with Tsavkelova et al.31, who reported that the strains of Rhizobium are among the most active IAA producers. Similarly Mandal et al.32 reported that Rhizobia were the first group of bacteria, which are attributed to the ability of PGPR to release IAA.
The present study tested the in vitro phosphate solubilizing capacity of 17 bacterial isolates comprising of genus Rhizobium, Bacillus, Paenibacillus and Pseudomonas. Sixteen of the 17 isolates were able to solubilize the insoluble tricalcium phosphate by the formation of the halo zones. The size of the solubilization varied among the bacteria isolates tested. Similarly, Andrade et al.33 reported a wide variations in solubilization indexes and zones among the tested isolates. The solubilization zone occurs due to the presence of some substances, such as organic acids, that are released by microorganisms into the medium that can form metal complexes with calcium, and thereby solubilize the P34. All the Bacillus strains tested in this study were able to solubilize the phosphate confirming what has been reported by previous studies indicating that they are efficient P solubilizers. For instance Andrade et al.33, indicated that isolates of the genus Bacillus sp. were the most frequent P solubilizers and classified them as high efficiency solubilizers. Bacillus megaterium M510 was found to solubilize both aluminium phosphate and iron phosphate in addition to moderate the solubilization of tri-calcium phosphate35. Das et al.36 indicated that Bacillus species isolated from rice rhizosphere solubilized phosphates, which was consistent with the results of our research for the isolate B. megaterium.
In addition to their beneficial nitrogen fixing activity with legumes, rhizobia can improve plant P nutrition by mobilizing inorganic and organic P. The present in vitro study showed that all the Rhizobium species were able to form solubilization zones with the tricalcium phosphate. Notably, the present results showed that Rhizobium pusense recorded significantly highest solubilization efficiency and solubilization index compared to the Bacillus strains. Earlier studies have also shown Rhizobium as efficient P solubilizers37,38,39.
Results from the present study showed that inoculation with Rhizobium strains significantly affected the nodulation of common bean. This is in agreement with what has been previously reported by other authors. For instance Bastos40 showed that inoculation with efficient rhizobial isolates promoted nodulation in common bean. The rhizobia strains used in this study were shown to have high IAA producing efficiency, thus resulting in enhanced nodule number and weights compared to the uninoculated control. Gosh et al.12 reported that the number of effective nodules were increased when inoculated with IAA-producing rhizobia in Cajanus cajan. Similarly, Pii et al.41 showed that IAA producing Rhizobium strains led to increased nodulation.
Inoculation of the four Rhizobium strains and their co-inoculation with the three PSB generally increased nodules compared with the uninoculated control. Co-inoculation of common bean with rhizobia and PSB led to an increase in nodule number and nodule dry weight compared to the single rhizobia inoculation. This could be attributed to the multi-strain’s ability to effectively nodulate and enhance solubilization of other essential soil minerals such as phosphorus42. Similar stimulatory effects on nodulation by co-inoculation of rhizobia and PSB has been reported by other authors43,44,45.
On the other hand, the present study suggested that the coinoculation of Rhizobium and the PSB might not always increase nodules compared with the individual inoculation with one of the four Rhizobium strains. This results have been reported previously in white clover by46, who showed that the co-inoculation of CHB1120 and G31 significantly increased nodules of white clover compared with the individual inoculation of CHB1120, but the co-inoculation of CHB1121 and two PGPR significantly decreased nodules in comparison with the individual inoculation of CHB1121. This suggests that the compatibility between these two kinds of microorganisms should be evaluated before application.
Results from this study showed that common bean in pots inoculated with either single P. polymyxa or B. aryabhattai developed nodules. Other than rhizobia, it was expected that the other bacterial strains will not elicit nodule formation. However, over the years, a vast number of bacteria other than rhizobia have been found in nodules47,48,49. A review by50 highlighted that some of these non-rhizobial nodule endophytes have nif and nod genes and elicits nitrogen fixing nodules on nodules just like the rhizobia. A study by51 reported that the diazotrophic bacteria used in their study were found to have the nitrogen fixing genes and nodulated and enhanced nodulation in chickpea plants under greenhouse conditions. There is therefore a need to test more of the non-rhizobial nodule endophytes for their nitrogen fixing ability and presence of nod genes to further understand their mechanisms of plant growth promotion.
Results from the present study showed that inoculation of common bean with rhizobia generally increased the shoot biomass and root dry weight compared to the control. The improved growth of plants subjected to Rhizobium inoculation is effectively attributed to its positive effect due to the symbiotic relationship between the rhizobia and the common bean52. Inoculation of seeds by phosphate solubilizing microorganisms is known to improve solubilization of insoluble phosphorus, which can therefore increase plant growth by enhancing the symbiotic efficiency of the common bean53,54.
In the present study, combined inoculation of the common bean with the rhizobia and PSB resulted in higher shoot and root dry weights compared to the single rhizobia inoculation. This can be attributed to better establishment of Rhizobium-legume symbiosis due to more secretion of plant growth promoting hormones, and improved nutrient availability especially P53,55. Similarly, Kumar et al.56 reported a growth enhancement of common bean by application of Bacillus and their combination with Rhizobium. Khalifa and Almalki57 showed that co-inoculation of phosphate-solubilizing B. megaterium and Sinorhizobium meliloti had a positive effect on the growth of common bean. Co-inoculation of Rhizobium MAP7 along with Brevibacillus MAP4 significantly increase the shoot dry weight compared to the treatment with Rhizobium MAP7 alone58.
Similar to the results on nodulation, some of the co-inoculation did not lead to improved growth as compared to the single inoculation. For instance, the shoot biomass of common bean co-inoculated with R. pusense and B. megaterium was significantly lower compared to their individual inoculations. This suggest that the two strains were not compatible. Other studies have shown the lack of positive effects of co-inoculation in respect to single rhizobium inoculation59,60. Therefore, compatibility studies should be done before coming up with the right microbial consortia for formulations of biofertilizers to ensure enhanced crop growth and maximum benefits from the plant growth promotion of the introduced microorganisms.
Results from this study revealed that inoculation of the common bean with Rhizobia strains increased the shoot N content compared to the uninoculated control. This could be attributed to the formation of nodules by the Rhizobium that stimulated biological nitrogen fixation by the crop. Similarly, de Souza et al.61 reported increased shoot N concentrations by Rhizobium when common beans were inoculated with R. leguminosarum strains. Similarly, shoot P concentration was increased as a results of inoculation with the PSB and Rhizobia. Earlier study by Chen et al.62 showed that the use of phosphate solubilising bacteria as inoculants increases the P uptake by plants. These findings are similar to the study by Neila et al.63 who observed that native rhizobia increase shoot phosphorus in bean. The present study showed a stimulatory effect in specific Rhizobia-PSB interactions in the total N and P concentration in the plant tissues. For example, R. phaseoli + B. aryabhattai co-inoculation had significantly higher N concentration compared to the single R. phaseoli inoculation. The enhancement in total N and P content of shoot in present study might be due to increase of nitrogen and phosphorous acquisition due to altering root structure and nodule formation in the crop64. Co-inoculation of common bean with Rhizobium and Bacillus strains was shown to improve nitrogen and phosphorus content compared to single Rhizobium inoculation65. A study by Nimnoi et al.66 showed that the total N and P content of shoot was enhanced by co-inoculation of Nocardia alba strain S4301 with Bradyrhizobium japonicum USDA110 as compared to single inoculation of Nocardia alba strain S4301 and un-inoculated control treatments in soybean. The increased N content from the co-inoculation of Bacillus and Rhizobia strains could also be attributed to the nitrogen fixing ability of the Bacillus. A study on nitrogen fixing potential of diverse species of Bacillus has reported the presence of nifH gene and hence the capability to fix atmospheric nitrogen67,68.
Native soil bacteria possesses ability to produce indole acetic acid growth hormones and to solubilize insoluble phosphate. Combined inoculation of rhizobia and PSB promoted the growth of common bean. Therefore, the phosphate solubilizing strains and the nitrogen fixing bacterial strains have great potential in being formulated and used as biofertilizers that can be tested under varying field conditions. This study highlights the importance of the use of phosphate solubilizing and IAA producer microorganisms as biofertilizers to enhance common bean growth.