Cowpea (Vigna unguiculata L. Walp) hosts several widespread bradyrhizobial root nodule symbionts across contrasting agro-ecological production areas in Kenya

Abstract

Cowpea (Vigna unguiculata L. Walp.) is an important African food legume suitable for dry regions. It is the main legume in two contrasting agro-ecological regions of Kenya as an important component of crop rotations because of its relative tolerance to unpredictable drought events. This study was carried out in an effort to establish a collection of bacterial root nodule symbionts and determine their relationship to physicochemical soil parameters as well as any geographical distributional patterns. Bradyrhizobium spp. were found to be widespread in this study and several different types could be identified at each site. Unique but rare symbionts were recovered from the nodules of plants sampled in a drier in-land region, where there were also overall more different bradyrhizobia found. Plants raised in soil from uncultivated sites with a natural vegetation cover tended to also associate with more different bradyrizobia. The occurrence and abundance of different bradyrhizobia correlated with differences in soil texture and pH, but did neither with the agro-ecological origin, nor the origin from cultivated (n = 15) or uncultivated (n = 5) sites. The analytical method, protein profiling of isolated strains by Matrix-Assisted Laser Desorption Ionization Time-of-Flight Mass Spectrometry (MALDI-TOF MS), provided higher resolution than 16S rRNA gene sequencing and was applied in this study for the first time to isolates recovered directly from field-collected cowpea root nodules. The method thus seems suitable for screening isolate collections on the presence of different groups, which, provided an appropriate reference database, can also be assigned to known species.

Keywords: Agro-ecology; Bradyrhizobium distribution; Cowpea (Vigna unguiculata L. Walp); MALDI-TOF MS.

Fig. 1

Dendrogram of an unsupervised hierarchical cluster analysis of rhizobial strains isolated from cowpea nodules based on Dice distances of mass spectral protein profiles of Matrix Assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometry (MS). Presence/absence of protein masses in the size range of mass-to charge ratio (m/z) 3000–12,000 was used. Strain origin is indicated in the strain identifiers (K: Kilifi, M: Mbeere). The strains 1–15 are from cultivated sites and the strains 16–20 from uncultivated sites. Reference strains were the isolates CBA from commercial Biofix inoculum (MEA Ltd. Nakuru, Kenya) and BK1 from a trap culture with soils from Burkina Faso. The scale bar shows the Dice distance in per cent.

Fig. 2

Maximum-likelihood phylogenetic tree based on near full-length 16S rRNA gene sequences of 25 Bradyrhizobium strains isolated from root nodules of cowpea and 17 species type strain sequences from the public databases. The tree is rooted with a further public database sequence of a type strain of a species of the genus Rhizobium. The statistical branch support values were obtained by bootstrap analysis (>50%) of 1000 resampled datasets. The strain identifiers indicate the geographical origin (K: Kilifi, M: Mbeere agroecological region). The strains 1–15 are from cultivated sites and the strains 16–20 from uncultivated sites. The sequence accession numbers are given in parentheses and the scale bar indicates the percentage of nucleotide substitutions. The colour coding follows that used in the dendrogram of Fig. 1.

Fig. 3

(a) Frequency of occurrence of five Bradyrhizobium isolate groups from root nodules of cowpea at cultivated and uncultivated sites in the agro-ecological regions around Mbeere and Kilifi in Kenya. The isolate grouping is based on similarity clustering of mass spectral protein profiles as determined by Matrix Assisted Laser Desorption/ Ionization Time of Flight (MALDI-TOF) Mass Spectrometry (MS) (Fig. 1). Sampling took place at 15 cultivated and 5 uncultivated sites in each agro-ecological region. (b) Rarefaction curves of the number of different groups of Bradyrhizobium in the two agro-ecological regions and cultivated and uncultivated sites (Fig. 1). Number of isolates considered: Mbeere, cultivated: 66, uncultivated: 12; Kilifi, cultivated: 68, uncultivated: 25.

Fig. 4

Correlative relationship between the occurrence of five similarity clusters of Bradyrhizobium root nodule isolates of cowpea and edaphic parameters as inferred by redundancy analysis (RDA). The biplot explains 96.3% of total variance in the dataset and is based on the presence/absence data of five similarity clusters of 156 Bradyrhizobium isolates, recovered from root nodules of cowpea plants growing in soil of 36 field sites. The vector sizes denote the strength of correlation and small angles indicate high correlation between environmental factors and/or cluster occurrence. Black arrows denote soil parameters, and blue arrows the Bradyrhizobium clusters, as characterized by similarity of MALDI-TOF MS protein mass spectra (Fig. 1, Fig. 3a). Percentages on the axes show the fraction of total variance explained. Only environmental parameters significantly correlated to the occurrence of the Bradyrhizobium clusters are shown. The pH (P < 0.05***), sand (P < 0.01**), and clay (P < 0.001*) concentrations, were those factors, which influenced Bradyrhizobium occurrence and symbiotic abundance. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)