ASR血rata Applied Soil Ecology 143 (2019)26-34 ed with holcus lanat (eu inity in soil under ryegrass (grass)and MF MS CF CS rial com e高g nunity are shaped a ing to the pattern of root exu of the n the ere n plan osphere+2 devel e V4 region erlgroecosystemsudiesabouthg ain Reaction (PCR)reaction volume ned the fo Mainly for cowpea,theres about the structure of the Madison SA),5.0pL of buffe 5x (Mg L0 unit of platinum community addin ab rhi ect and the diver ty and structure of nd r 30s,with a final extension of 3 min at 72'C to ensure (2)h this study g,the PCR products ed up using Agencour A).ac ding t the fc factur 2.Material and methods 2.1.Experimental site and soil sampling ermined and d then nt was conducted at the De tofsoilscicnce f8.0 soil. he exr ntal design was con data we plot p d using QIME following the UPARSE ive plants m ys. eipictedandn3etooig" ered into OTUs at a %6 similarity cut-off folle amed MF)and 75 (se. 81 2. (bu soil)T and with soil ver 22.DNA extraction and sequencing or th Monte carlo pe tion test(legume) when compared with Holcus lanatus (graminea) (Ladygina and Hedlund, 2010). Recently, Zhou et al. (2017) have found differences between the bacterial community in soil under ryegrass (grass) and alfalfa (legume). The plant developmental stage is another factor influencing the bacterial community in the rhizosphere (Mougel et al., 2006). During the plant growth, the roots exudation changes according to the stage of development, and the abundance and structure of the rhizospheric microbial community are shaped according to the pattern of root exu￾dation that may vary from young seedling, flowering, to plant senes￾cence (Dunfield and Germida, 2003). Indeed, Houlden et al. (2008) evaluated the response of the microbial community to the different growth stage of pea, wheat and sugar beet and observed changes in the bacterial community following the developmental stage for all the tested plants. Considering the crucial role of the rhizosphere micro￾biome for plant growth and health, it is essential to elucidate how different plant species at different growth stages select differently the microbial community inhabiting their rhizosphere. Maize (grass) and cowpea (legume) are two important plant species cultivated in the world being used for human and animal feeding and cover crop (Ramirez-Cabral et al., 2017; Boukar et al., 2016). Although these plants are distributed in several agroecosystems, studies about the bacterial community in the rhizosphere of these species are scarce. Mainly for cowpea, there is no information about the structure of the bacterial community in the rhizosphere. Also, it is unclear the effect of the different stage of development in these plants on the soil bacterial community. In order to determine the extent to which different plant species are able to select a distinct rhizospheric microbiome, in this study we hy￾pothesized that (1) maize and cowpea, as different plant species, exert different rhizosphere effect and shape the diversity and structure of bacterial community differently; and (2) the soil bacterial community changes during the flowering and senescence of plants. Thus, this study aimed to evaluate the diversity and structure of the bacterial commu￾nity in the rhizosphere of maize and cowpea during the flowering and senescence using the 16S rRNA sequencing. 2. Material and methods 2.1. Experimental site and soil sampling The experiment was conducted at the Department of Soil Science from the Federal University of Piauí, Brazil. The soil of the experimental site is classified as Fluvisol soil. The experimental design was com￾pletely randomized with three replicates. Each experimental plot pre￾sents 20 m2 (12 m2 of usable area). Maize (Zea mays L.) was sowed at a density of five plants m−1 and was grown for 75 days. Afterward, cowpea [Vigna unguiculata (L.) Walp.] was sowed at the density of six plants m−1 and was grown for 65 days. To measure the rhizospheric influence of each plant species on the bacterial community, soil samples adhered to the roots were collected and mixed to form a composite sample in each plot. During maize growth, soils were collected at 45 (flowering, named MF) and 75 (se￾nescence, MS) days. During cowpea growth, the sampling occurred at 35 (flowering, CF) and 65 (senescence, CS) days. Soil samples without plants effects were collected as control (bulk soil). The soil samples were sieved (2-mm) and stored at −20 °C. Soil chemical and physical properties, estimated according to Embrapa (1997), are shown in Table 1. 2.2. DNA extraction and sequencing Total DNA was extracted from 0.5 g (total humid weight) of soil using the PowerLyzer PowerSoil DNA Isolation Kit (MoBIO Laboratories, Carlsbad, CA, USA), according to the manufacturer's in￾structions. The DNA extraction was performed in triplicate for each soil sample, totalizing 15 samples [(bulk soil + 2 rhizosphere + 2 devel￾opmental stage) × 3 replicates]. The V4 region of the 16S rRNA gene was amplified with region￾specific primers (515F/806R) (Caporaso et al., 2011). Each 25 μL Polymerase Chain Reaction (PCR) reaction volume contained the fol￾lowing: 12.25 μL of nuclease-free water (Certified Nuclease-free, Pro￾mega, Madison, WI, USA), 5.0 μL of buffer solution 5× (MgCl2 2 Mm), 0.3 mM dNTP's 0.3 μM of each primer (515 YF 40 μM e 806 R 10 μM), 1.0 unit of Platinum Taq polymerase High Fidelity in concentration of 0.5 μL (Invitrogen, Carlsbad, CA, USA), and 2.0 μL (10 ng μL−1 ) of template DNA. Moreover, a control reaction was performed by adding water in place of DNA. The conditions for PCR were as follows: 95 °C for 3 min to denature the DNA, with 35 cycles at 98 °C for 20 s, 55 °C for 20 s, and 72 °C for 30 s, with a final extension of 3 min at 72 °C to ensure complete elongation. After indexing, the PCR products were cleaned up using Agencourt AMPure XP – PCR purification beads (Beckman Coulter, Brea, CA, USA), according to the manufacturer's manual, and quantified using the dsDNA BR assay Kit (Invitrogen, Carlsbad, CA, USA) on a Qubit 2.0 fluorometer (Invitrogen, Carlsbad, CA, USA). Once quantified, an equimolar concentration of each library (50 ng) was pooled into a single tube. After quantification, the molarity of the pool was de￾termined and diluted to 2 nM, denatured, and then diluted to a final concentration of 8.0 pM with a 20% PhiX (Illumina, San Diego, CA, USA) spike for loading into the Illumina MiSeq sequencing machine (Illumina, San Diego, CA, USA). Sequence data were processed using QIIME following the UPARSE standard pipeline according to Brazilian Microbiome Project (Pylro et al., 2014), to produce an OTU table and a set of representative se￾quences. Briefly, the reads were truncated at 240 bp and quality-filtered using a maximum expected error value of 0.5. Pre-filtered reads were dereplicated, and singletons were removed and filtered for additional chimeras using the RDP_gold database using USEARCH 7.0. These se￾quences were clustered into OTUs at a 97% similarity cut-off following the UPARSE pipeline. After clustering, the sequences were aligned and taxonomically classified against the Greengenes database (version 13.8). 2.3. Statistical analysis The community structure and its correlation with soil parameters were visualized using Redundancy analysis (RDA). All matrices were initially analyzed using Detrended Correspondent Analysis (DCA) to evaluate the distribution of the data, revealing that the best-fit math￾ematical model for the data was RDA. Forward selection (FS) and the Monte Carlo permutation test were applied with 1000 random per￾mutations to verify the significance of environmental parameters upon the microbial community structure. RDA plots were generated using Table 1 Chemical and physical properties of soil. BS MF MS CF CS pH (water) 5.6 6.3 6.7 6.2 6.0 EC (dS m−1 ) 0.3 0.5 0.7 0.5 0.5 TOC (g kg−1 ) 5.9 5.9 6.7 5.4 5.6 P (mg kg−1 ) 5.5 5.8 6.0 4.8 4.3 K (mg kg−1 ) 70 89 85 81 78 Ca (mg kg−1 ) 213 317 328 320 296 Mg (mg kg−1 ) 38 61 64 61 58 Na (mg kg−1 ) 69 98 93 85 91 Sand (%) 60 60 60 60 60 Silt (%) 28 28 28 28 28 Clay (%) 12 12 12 12 12 BS – Bulk soil; MF – maize flowering; MS – maize senescence; CF – cowpea flowering; CS – cowpea senescence; TOC – total organic C; EC – electric con￾ductivity. A.S.F. de Araujo, et al. Applied Soil Ecology 143 (2019) 26–34 27