Appl Microbiol Biotechnol https://doi.org/10.1007/s00253-017-8550-8 GENOMICS, TRANSCRIPTOMICS, PROTEOMICS Interactions of plant growth-promoting rhizobacteria and soil factors in two leguminous plants Xiao Xiao1 & Miaochun Fan1 & Entao Wang2 & Weimin Chen1 & Gehong Wei1 Received: 13 July 2017 /Revised: 8 September 2017 /Accepted: 17 September 2017 # Springer-Verlag GmbH Germany 2017 Abstract Although the rhizomicrobiome has been extensively Group 4 were significantly correlated with soil N, K and C. The studied, little is known about the interactions between soil prop- influence of soil properties on the relative abundance of plant erties and the assemblage of plant growth-promoting microbes in biomass-associated rhizomicrobes differed between soybean the rhizosphere. Herein, we analysed the composition and struc- and alfalfa. The results suggest the rhizomicrobiome has a pro- ture of rhizomicrobiomes associated with soybean and alfalfa nounced influence on plant growth, and the rhizomicrobiome plants growing in different soil types using deep Illumina 16S assemblage and plant growth-associated microbes are differen- rRNA sequencing. Soil pH, P and K significantly affected the tially structured by soil properties and leguminous plant species. composition of the soybean rhizomicrobiome, whereas soil pH and N had a significant effect on the alfalfa rhizomicrobiome. Keywords Plant growth-promoting rhizobacteria . Illumina Plant biomass was influenced by plant species, the composition sequencing . Plant-microbial interactions . Rhizosphere . of the rhizomicrobiome, soil pH, N, P and plant growth stage. Leguminous plants The beta diversity of the rhizomicrobiome was the second most influential factor on plant growth (biomass). Rhizomicrobes as- sociated with plant biomass were identified and divided into four groups: (1) positively associated with soybean biomass; (2) neg- Introduction atively associated with soybean biomass; (3) positively associat- ed with alfalfa biomass; and (4) negatively associated with alfalfa The rhizosphere, the soil in the immediate vicinity of plant biomass. Genera assemblages among the four groups differen- roots, is strongly influenced by both the roots and the sur- tially responded to soil properties; Group 1 and Group 2 were rounding soil (Lundberg et al. 2012) and involves an enor- significantly correlated with soil pH and P, whereas Group 3 and mous interactions between the roots, soil and microbes. Plants release up to 40% of their photosynthetically fixed car- Electronic supplementary material The online version of this article bon below ground (Hutsch et al. 2002;Staff2015), of which (https://doi.org/10.1007/s00253-017-8550-8) contains supplementary ~ 11% is retained in rhizodeposition (Jones et al. 2009). The material, which is available to authorized users. rhizodeposition provides a constant flow of plant-based or- ganic substrates to soil microbes colonising the rhizosphere * Weimin Chen (Kai et al. 2016). In return, rhizobacteria play a role in pro- [email protected] moting or inhibiting plant growth (Kai et al. 2016). Some * Gehong Wei rhizobacteria, so-called plant growth-promoting rhizobacteria [email protected] (PGPR), stimulate plant growth indirectly or directly through different mechanisms. For example, they can increase the ab- 1 State Key Laboratory of Crop Stress Biology in Arid Areas, College of Life Sciences, Northwest A&F University, sorption of mineral nutrients (Vacheron et al. 2013), stimulate Yangling 712100, Shaanxi, People’sRepublicofChina the synthesis of phytohormones and secondary metabolites 2 Departamento de Microbiología, Escuela Nacional de Ciencias (Gamalero and Glick 2015), suppress phytopathogens and Biológicas, Instituto Politécnico Nacional, 11340 México, D.F., enhance plant tolerance to environmental stress (Anderson Mexico and Habiger 2012). Appl Microbiol Biotechnol PGPR comprise a range of species with huge taxonomic di- (Chaparro et al. 2014). The interplay of all these factors (plant, versity, especially within Firmicutes and Proteobacteria soil and rhizobacteria) makes the rhizosphere a complex and (Vacheron et al. 2013; Vessey 2003). Bacillus, Pseudomonas, dynamic microecosystem that requires further exploration. Escherichia, Micrococcus and Staphylococcus have been con- And yet, the effects of soil type, plant species and growth firmed as producers of auxins (Ali et al. 2010; Zhang et al. 2007), stages on PGPR have not been comprehensively studied. and Pseudomonas is a producer of 2,3-diacetylphloroglucinol, an Herein, we performed a deep Illumina sequencing of 16S antimicrobial compound (Picard et al. 2000). Azospirillum rRNA amplicons to reveal the rhizomicrobiome composition brasilense can produce nitric oxide (Creus et al. 2005), a signal- of two plant species (soybean and alfalfa) grown in three soil ling molecule modulating plant physiological responses and types (chernozem, cinnamon and red soils). In order to inves- influencing root growth and developmental processes tigate the relationships among plants (biomass, growth stage (Vacheron et al. 2013). Pseudomonas, Bacillus and Rhizobium and species), soil properties and the rhizomicrobiome, we test- areabletoincreasethenutrient absorption of plant roots by ed the following: (1) whether plant biomass is associated with dissolving insoluble phosphate (Goldstein 1995; Hariprasad the composition of the rhizomicrobiome; (2) what kind of and Niranjana 2009). Additionally, many rhizobacteria can fix rhizomicrobes was positively or negatively correlated to plant N2 and produce extracellular siderophores that improve plant growth in soils with different properties; and (3) which soil growth (Cakmakçi et al. 2006; Kloepper et al. 1980). PGPR also properties determine the rhizomicrobe species correlated with are able to enhance plant tolerance to environmental stresses, plant growth. This design allowed us to gain a better under- such as salinity (Tank and Saraf 2010), drought (Dimkpa et al. standing of the relationship between the rhizomicrobes and 2009) and heavy metals (Dell’Amico et al. 2005). Most previous plant growth by combining soil parameters, plant species and studies on PGPR involved culture-based methods, such as plant growth stages. The results may assist in further utilising screening bacteria for the production of antibiotics, siderophores the productive promoting communities in field conditions. and phytohormones (Anderson and Habiger 2012). However, culture-based techniques can only explore a small proportion of rhizomicrobiome species, while most species are overlooked Materials and methods (Anderson and Habiger 2012). By contrast, the Illumina se- quencing of 16S rRNA amplicons could help to elucidate the Sampling strategy and data collection rhizomicrobiome species more comprehensively. The composition of the rhizomicrobiome is strongly affect- We collected farmland soil samples representing three major ed by the soil and plants (Lundberg et al. 2012; Peiffer et al. soil types in China: chernozem (CH; Mollisol) from 2013). Significant variations were found in the composition of Heilongjiang Province (46° 24′ 10.2″ N, 125° 21′ 59.5″ E), maize and Arabidopsis rhizomicrobiomes in different soil cinnamon soils (CI; Alfisol) from Shaanxi Province (34° 4′ types using a metagenomic approach (Lundberg et al. 2012; 14.88″ N, 108° 36′ 8.28″ E) and red earth (RE; Acrorthox) Peiffer et al. 2013). Specific soil properties such as pH, nitro- from Jiangxi Province (28° 21′ 41.5″ N, 115° 55′ 0.80″ E). gen (N), phosphorus (P), potassium (K) and other mineral Prior to soil sampling, the litter layer was removed. Surface nutrients exhibit different effects on the composition of the soil samples (0–20 cm) were collected with a sterilised shovel rhizobacterial community (Berg and Smalla 2009;Edwards using the five-point sampling method as previously described et al. 2015;Lundbergetal.2012; Pii et al. 2016; Schreiter (Navarrete et al. 2015). Soil samples were transported imme- et al. 2014). In three California grasslands, the composition of diately to the laboratory and stored at 4 °C in the dark. All the wild oat rhizomicrobiome was shown to be significantly samples were passed through a sterile 2 mm sieve before use. correlated with soil pH and K (Nuccio et al. 2016). Soil pH, as Two plant species, soybean (grain legume) and alfalfa (for- well as K, P, cadmium and magnesium levels, was significant- age legume), were used in this study. After surface sterilisation ly correlated with the relative abundance of Proteobacteria in of seeds (Edwards et al. 2015), soybean (three plants per pot) the rhizosphere of Japanese barberry (Coats et al. 2014). and alfalfa (eight plants per pot) were grown in pots contain- It is believed that specific microbial populations associated ing homogenised soil (CI, CH or RE) under a 16-h light with plant roots are selected by their unique root exudates (25 °C) and 8-h dark (20 °C) cycles with a relative humidity (Berg and Smalla 2009), which provide nutrients for microbes of 40 to 45%. To ensure sufficient sample for further analysis, and contain antimicrobial metabolites. Through qualitative rhizosphere soil from two pots were combined as one repli- and quantitative differences of root exudates, some plant spe- cate. A total of 162 pots [two legume species × three soils × cies can selectively modulate the composition of the three periods × six pots + three soils × three periods × six pots rhizomicrobiome (Pii et al. 2016). Plant growth stage also (without