Soybeans provide abundant protein and oil for human and animal diets. During soybean growth, nitrogen plays a critical role and is demanded in a great amount (Freitas et al., 2022; Mayhood et al., 2021). Fortunately, soybeans can form symbiotic associations with rhizobia as nodules to fix atmospheric N2 into ammonia (Cbl et al., 2019; Mariana et al., 2023). Successful rhizobia–legume symbioses are the most important nitrogen (N) source in agroecosystem, depending on efficient plant root nodulation and subsequent N2 fixation (Ferguson et al., 2018). Currently, the amount of symbiotically fixed N2 totals roughly 50%-60% of that demanded in soybean lifetime (Roy et al., 2019). This natural process in rhizobia–legume symbioses is of vital importance in reducing the application of chemical fertilizers and providing more clear nitrogen source for soybean development.
Root hair is the initial infection site for symbiotic (rhizobia) in soybean as well as other legume plants (Levine et al., 2007; Cervantes-Pérez et al., 2022; Chao et al., 2023). During nodule formation, rhizobia bacteria always attach to root hairs, induce root hair deformation, and form infection thread (Gage et al., 2000; María et al., 2022). Subsequently, the infection thread induces the bacteria to the root cortex and ultimately forms nodule organs with the ability to fix N2 (Nishida et al., 2018; Wang et al., 2019). The inoculation of rhizobia was closely related to some changes in root morphology and structure (Li et al., 2015; Yang et al., 2017; Soyano et al., 2019; Schiessl et al., 2019). Li (Li et al., 2015) found that soybean rhizobia increased the size and number of cortical cells in root meristem and elongation areas, which expanded root hair density and root hair area and enable more nodulation. The deformed root hairs always serve as entrapping attached bacteria in the rhizobia–legume symbioses, and have been widely reported to be many shapes such as curling, wiggling, branching, and shepherd (Esseling, et al., 2003; Hwang et al., 2014; Ke et al., 2016; Velandia et al., 2022). Soybean root hair deformation responded in increase to rhizobia (nod factor) concentration (Duzan et al., 2004), but whether more deformed root hairs lead to more nodulation is not clear by literature. In addition, little knowledge is available on the process of soybean root hair deformation, nodule initial, and nodule development over time in situ.
Therefore, it is very important to study on root hair deformation progress in soybean-rhizobia symbioses, which will help to understand the process in root hair deformation mechanism. Root hairs are very tiny and sensitive part in rhizobia–legume symbiosis (Fournier J et al., 2008; et al., 2021). The length of root hairs ranges from tens of microns to hundreds of microns, but only dozens of microns in width. Owing to the difficulty in observing roots into soil, most researches were carried with destructive method or in agar/solution environment observed using light microscopy (Zhaoming et al., 2017; Schiessl et al., 2019; Xue et al., 2023). It either had the limitation of losing root hairs during sampling, or could not represent real growth under soil environment. In addition, these methods didn’t support successive studies on the same roots over time. X-ray computed tomography (X-CT) (Scotson et al., 2021), nuclear magnetic resonance (NMR) (Metzner et al., 2015) and electrical impedance tomography (EIT) (Peruzzo et al., 2021) were not intended for such tiny root hair traits. To make it applicable to focus on detailed roots such as root hairs, rhizotrons and minirhizotrons were improved and adapted to a smaller scale by amplifying local area (Cai et al., 2016; Amato et al., 2012; Lu et al., 2019; Lu et al., 2022).
In this study, we intended (1) to observe root hair traits and root nodulation development in rhizobia–legume symbiosis over time with previous designed microrhizotron (1.5 cm3 in volume) in situ, (2) to clarify detailed root hair deformation from the aspect of shapes and curling angle (deforming extent), (3) to analyze relationship between root hair deformation and nodulation (number/volume), thus to comprehend mechanism in the process of root hair deformation and nodulation, and provide a method for the screening of soybean-rhizobia genotypes with higher nitrogen fixing ability