Comment: Isolation and Screening of Bacteria for Their Diazotrophic Potential and Their Influence on Growth Promotion of Maize Seedlings in Greenhouses

Maize Seedlings in Greenhouses. Recent papers by Kifle and Laing showed that diazotrophs isolated from Maize tissue and Maize-associated soil have the potential to promote growth of this important crop when used to inoculate seeds prior to planting (Kifle and Laing, 2016a,b). The fact that the authors focused their studies on diazotrophs presumes that the biological nitrogen fixation (BNF) is key to the mechanism of plant growth promotion by the microorganisms they tested. However, numerous other studies (e.g., Khalid et al., 2004) have found plant growth promotion by organisms isolated from the rhizosphere that were not screened for BNF, implying that this trait is not necessarily the key mechanism of plant growth promotion. By conducting additional analysis of their data, we find the striking result that rates of acetylene reduction in pure culture presented by Kifle and Laing (2016a) show a strong positive relationship with two metrics of plant health measured in their greenhouse experiment: chlorophyll content (Figure 1A) and stomatal conductance (Figure 1B). The acetylene reduction assay as conducted by Kifle and Laing (2016a) should serve as a proxy for maximum potential rates of BNF achievable by the diazotrophs they investigated. The relationships shown in Figures 1A,B therefore lend credence to the argument that BNF plays an important role in plant growth promotion by the microbes that Kifle and Laing (2016a) tested. By omitting fixed nitrogen sources from their fertilization regime, Kifle and Laing (2016a) designed their diazotroph inoculation experiment to investigate the direct role of BNF in plant growth promotion. Indeed, studies using stable isotope tracers have shown that nitrogen fixed by diazotrophic inoculants was incorporated into plant biomass. For example, Zakry et al. (2012) used the 15 N pool dilution technique to show that Oil Palm plants inoculated with the endophyte Bacillus sphaericus UPMB-10 obtained the majority of their nitrogen from fixation after 8 months of growth, while Knoth et al. (2014) found similar results for Poplar plants inoculated with multi-strain diazotrophic consortia. However, bacteria are known to promote plant growth through multiple other mechanisms besides diazotrophy (Friesen et al., 2011) and these mechanisms, which we outline below, may be at least partially responsible for the relationships presented in Figure 1 as well. Bacteria may increase nitrogen concentrations without diazotrophy through exoezyme release (e.g., Wallenstein and Weintraub, 2008). Rhizosphere bacteria may also enhance plant growth by altering the availability of nutrients other than nitrogen, such as through …