In plants, co-inoculation is performed with a combination of different bacterial genera or species with the aim to modify plant growth and development synergistically. A great number of non-symbiotic or free-living bacteria are known to promote symbiosis in legumes when co-inoculated with rhizobia. A recent analysis of over 300 cases of positive plant responses to Azospirillum in 12 countries (Cassán and Díaz-Zorita 2016) found that the rise in its co-inoculation with rhizobia led to a 6.6% increase in legume yield over rhizobia-only treatments. In the last decades, research in Argentina and Brazil has assessed the benefits of this kind of co-inoculation for the cultivation of soybean (Hungria et al. 2013; Hungria et al. 2015; Queiroz Rego et al. 2018; Deak et al. 2020; Moretti et al. 2020; Rondina et al. 2020). The combined use of rhizobia and Azospirillum for the growth of legumes, mainly soybean, goes back to the 1990s. Inoculants containing Bradyrhizobium and A. brasilense were authorized for commercial use on soybean in Brazil in 2013, and have shown to increase grain yields (Hungria et al. 2013; Hungria and Nogueira 2019; dos Santos et al. 2019). There are about eighty registered products in South America whose active agent is A. brasilense, with AbAz39 strain being the most frequent (Cassán et al. 2020). Moreover, in Argentina there are over 15 commercial inoculants for soybean formulated based on BjE109 (the most widely recommended strain for inoculation), for which co-inoculation with A. brasilense is also advised. Currently, there is an increasing use of Azospirillum-based inoculants in Brazil soybean and beans (Phaseolus vulgaris). In the state of Parana, for instance, the use of co-inoculation rose by almost 30% between 2016 and 2018 (Prando et al. 2016; 2018). Despite the data available regarding commercial and agricultural performance, little is known about the exact reasons why this type of co-inoculation improves plant growth. And furthermore, if these responses are do to interactions between Azospirillum and rhizobia or because of the interaction of Azospirillum and the plants themselves.
In this study, with the aim of finding further evidence that may elucidate the relative importance of the soybean-Azospirillum and Bradyrhizobium-Azospirillum interactions, we evaluated the effect of co-inoculating soybean seeds with BjE109 and AbAz39 at different ratios and interaction times. Both, in vivo and in planta assays were carried out to compare the performance between co-inoculation and single inoculation treatments with the bacterial strains on their own. The co-inoculation, combining Bradyrhizobium and Azospirillum strains, in a 1:1 proportion when treating the soybean seeds, represents the frequent co-inoculation treatment applied at farmers’ fields. In this case, both strains are mixed and start their interaction with each other at the same time they are inoculated on the seeds. In this scenario, most of the effects of the presence of Azospirillum have direct impact on the plant. If the mix of microorganisms is prepared 24 hours before treating the seeds (T24), the strains interact with each other before reaching the seed coat and the derived benefits on nodulation and soybean growth could be interpreted from the effects of Azospirillum on Bradyrhizobium performance.
Because of its backbone contribution in the nitrogen nutrition of soybean plants, the main microorganism in the co-inoculation mixture is BjE109. Initially, to analyze the physiological behavior of BjE109, we assessed different proportions of the combination with Azospirillum (1:9, 1:1 and 9:1). The 1:1 treatment was chosen because it had the greatest values of EPS production, bacterial biomass, and IAA degradation for which AbAz39 is responsible (Table S1). These parameters were selected with reference to previous studies published by our laboratory (Torres et al. 2018; 2021), in which we confirmed that exogenous IAA addition to pure BjE109 cultures triggered physiological changes in the bacterium, such as the ability to increase its biomass and produce EPS, both are advantages on the rhizobia cell survival on soybean seeds and on its symbiotic performance. We also obsserved that BjE109 can degrade IAA when is added to the culture medium through the action of a 3-phenylpropanoate dioxygenase-like enzyme (subunit alpha and beta) within a cluster named iac (Torres et al. 2018; 2021). This must be relevant for a better performance of the co-inoculation so the mix at T24 doubled the EPS content and significantly increased the biomass production in comparison to the T0 treatment (Table 1). In turn, T0 outperformed inoculation with BjE109 alone. An increase in the production of EPS by rhizospheric bacteria like rhizobia is related to higher plant tolerance to stress caused by water, oxidation, low temperature, and other factors (Cytryn et al. 2007; Chang and Halverson 2003; Tamaru et al. 2005). The exposure of 1 mM IAA to B. diazoefficiens USDA110 increased the production of the EPS content, which also enhanced the plant tolerance to different types of stresses such as heat stress, cold stress, desiccation, among others (Donati et al. 2013).
When the BjE109 survival was evaluated, the co-inoculation allowed recovering of more viable cells compared to the rest of treatments, being better in the T24 mix than in T0 (Table 2). The recovery kinetics for BjE109 on soybean seeds, assessed over time and under the conditions imposed by the different treatments, agreed with our previous results on the greater recovery of BjE109 and B. diazoefficiens USDA110 cells in cultures pre-incubated with IAA (Torres et al. 2018; Donati et al. 2013). These findings suggest that the production of IAA by AbAz29 in the culture medium of the mix may be partly responsible for the increase in EPS, which may offer BjE109 a better chance at surviving on the seeds. Auxins have long been posited to play a major role in nodule ontogenesis within legume-rhizobium symbiosis (Thiman 1936), and there are many reports on alterations in nodule organogenesis due to changes in auxin content because of inoculation with auxin-producing bacteria or exogenous hormone addition (Schmidt et al. 1988). The co-inoculation with rhizobia and Azospirillum is not an exception. For example, co-inoculation with IAA-deficient Sinorhizobium meliloti and IAA-producing A. brasilense on alfalfa generated significantly more nodules on the primary root (Schmidt et al. 1988). Remans et al. (2008) also provided direct evidence on the enhancing effects of IAA by co-inoculating beans with R. etli and a mutant IAA-deficient A. brasilense. The literature, then, might lead us to believe that the improvement observed in legume growth after inoculation with Azospirillum is explained by a hypothetical interaction between the bacteria and the plant, in which phytohormone synthesis (primarily IAA) could be crucial.
The co-inoculation could also favor AbAz39 performance that was conditioned by the interaction time. In this regard, it has been observed that the interaction of Bradyrhizobium with Azospirillum in soybeans seeds highly improved drought tolerance (Rondina et al. 2020) or enhanced the growth and yield of soybean plants under arsenic stress (Armendariz et al. 2019). This must be because the co-inoculation increased the number of nodules on the roots, leading to higher nodule biomass and thus improved BFN (Torres et al. 2018; Rondina et al. 2020). We not only corroborated these results but also demonstrated that an interaction time of 24 h prior inoculation improved the establishment of symbiosis, BNF and, as consequence, plant development under controlled (greenhouse) and uncontrolled growth conditions (field) compared to T0, and much more when the bacteria are used individually (Table 4 and 5). However, we also demonstrate that the number but not the size of the nodule influences the quality and quantity of the plant yield (Fig. 3). In the last decades, several authors have analyzed the contribution of co-inoculation with Azospirillum sp. to legume productivity. In the pampas region (Argentina), 21 field trials performed on alfalfa showed that combined inoculation of Ensifer meliloti and A. brasilense was almost twice as effective as inoculation with rhizobia alone (Díaz-Zorita et al. 2012). Hungria et al. (2013) also reported increased yield for soybean and common bean when complementing seed inoculation with rhizobia with the in-furrow application of A. brasilense at four locations in Brazil. They found that inoculation of soybean with Bradyrhizobium resulted in an 8.4% increase in yield, against the 16.1% increase achieved by the combination of strains. For common beans, individual inoculation with R. tropici boosted yield by 8.3%, but the addition of A. brasilense raised this figure to 19.6%. A metadata analysis by Barbosa et al. (2021) revealed that soybean co-inoculation was related to a 2.8% increase in grain yield and a 3.2% increase in N concentration in the grains with respect to the inoculation only with Bradyrhizobium. In other 37 field trials, soybean co-inoculation with Bradyrhizobium and A. brasilense also increased mean yield by 227 kg ha−1 with respect to the inoculation with Bradyrhizobium alone and by 335 kg ha−1 with respect to the uninoculated control (Nogueira et al. 2018). Finally, co-inoculation of soybean was found to raise the nodulation percentage by around 5% in Brazil (Hungria et al. 2015; Fipke et al. 2016; Galindo et al. 2018) and around 12% in Argentina (Benintende et al. 2010; Ferraris and Couretot 2011, 2013; Morla et al. 2019), a difference which may be attributed to the tropical-subtropical and temperate conditions, respectively. Despite this, the limited dataset was insufficient to show a direct relationship between co-inoculation and changes in nodulation and grain yield.