High Temperatures and Bacillus Inoculation Affect the Diversity of Bradyrhizobia in Cowpea Root Nodules

Abstract

Future climatic scenario predictions indicate a substantial temperature increase, reducing crop production worldwide and demanding the development of adaptations in agriculture. This study aimed to assess the impact of high temperatures and amendments with Bacillus on nodulating bradyrhizobia. Two cowpea genotypes were evaluated at low (min = 20.0°C, max = 33.0°C) and high-temperature regimes (min = 24.8 C, max = 37.8°C). Plants were also inoculated with Bacillus sp. ESA 402, a plant growth-promoting bacterium. The molecular diversity of the bradyrhizobia isolated from cowpea nodules and plant growth was assessed. High temperatures reduced nodulation of the BRS Itaim cowpea genotype. One hundred and eighty-six were genotyped, clustering the collection into 45 groups. The high temperatures reduced the number of groups, but this negative influence was diminished by Bacillus inoculation. Alpha diversity showed little impact on the experimental interactions. However, this influence was evident for all factors and the interaction of the three factors when beta diversity was assessed. 16S rRNA and constitutive gene sequences identified all strains as Bradyrhizobium spp. mainly within the B. japonicum supercluster. Cowpea-Bradyrhizobium association diversity is multifactorial under different temperature regimes, as is the presence or absence of the plant-growth-promoting bacteria Bacillus sp. ESA 402.

Keywords: Bacillus sp; Bradyrhizobium; climate change; heat stress; inoculant.

Figure 1

Interaction of plant genotype (BRS Acauã and BRS Itaim) and inoculation of Bacillus sp. ESA 402 (inoculated or non‐inoculated) cowpea' shoot dry mass (n = 6). * = p < 0.05.

Figure 2

Influence of the air temperature as a single factor (n = 12) (A) or the interaction between the temperature and the cowpea genotype (BRS Acauã and BRS Itaim) (B, n = 6) on the number of nodules per plant. * = p < 0.05, ** = p < 0.01.

Figure 3

Different BOX‐PCR profiles by air temperature (A) and by the interaction between the air temperature (20.0–33.0°C or 24.8–37.8°C) and the genotype (BRS Acauã and BRS Itaim) (B), n = 6). * = p < 0.05.

Figure 4

Shannon‐Wiener (A) and Simpson (B) indexes based on the occurrence and abundance of 45 different BOX‐PCR genetic profiles. Indexes calculated by the interaction of the inoculation of Bacillus sp. ESA 402 (inoculated or non‐inoculated) and the interaction between the air temperature (20.0–33.0°C or 24.8–37.8°C) (n = 6). ° = p < 0.1.

Figure 5

Principal coordinates analysis (PCoA) by bacterial beta diversity among different factors and their interactions determined through PERMANOVA based on the Bray–Curtis distance matrix.

Figure 6

Maximum‐likelihood phylogenetic tree of the 16S rRNA gene sequences of 45 Bradyrhizobium strains from Vigna unguiculata root‐nodules plus 30 type strains (917 nucleotides). The numbers in the branches are the bootstrap values > 50% (1000 replications). Bosea thiooxidans DSM 9653 ^T was included as an outgroup. “ESA” strains are those from the current study and are shown in boldface with their GenBank accession number in parenthesis. Jukes‐Cantor model was used for phylogenetic reconstruction.

Figure 7

Maximum‐likelihood phylogenetic tree based on the concatenated sequence data for the genes (recA, gyrB, and rpoB) of 45 Bradyrhizobium strains and 30 type strains (1171 nucleotides) isolated from root nodules of Vigna unguiculata. The numbers in the branches are the bootstrap values > 50% (1000 replications). Bosea thiooxidans DSM 9653 ^T was included as an outgroup. “ESA” strains are those from the current study and are shown in boldface with their GenBank accession number in parenthesis. Jukes‐Cantor model was used for phylogenetic reconstruction.