Individual competence predominates over host nutritional status in Arabidopsis root exudate-mediated bacterial enrichment in a combination of four Burkholderiaceae species

Abstract

Background: Rhizosphere microorganisms play a crucial role in plant health and development. Plant root exudates (PRE) are a complex mixture of organic molecules and provide nutritional and signaling information to rhizosphere microorganisms. Burkholderiaceae species are non-abundant in the rhizosphere but exhibit a wide range of plant-growth-promoting and plant-health-protection effects. Most of these plant-associated microorganisms have been studied in isolation under laboratory conditions, whereas in nature, they interact in competition or cooperation with each other. To improve our understanding of the factors driving growth dynamics of low-abundant bacterial species in the rhizosphere, we hypothesized that the growth and survival of four Burkholderiaceae strains (Paraburkholderia phytofirmans PsJN, Cupriavidus metallidurans CH34, C. pinatubonensis JMP134 and C. taiwanensis LMG19424) in Arabidopsis thaliana PRE is affected by the presence of each other.

Results: Differential growth abilities of each strain were found depending on plant age and whether PRE was obtained after growth on N limitation conditions. The best-adapted strain to grow in PRE was P. phytofirmans PsJN, with C. pinatubonensis JMP134 growing better than the other two Cupriavidus strains. Individual strain behavior changed when they succeeded in combinations. Clustering analysis showed that the 4-member co-culture grouped with one of the best-adapted strains, either P. phytofirmans PsJN or C. pinatubonensis JMP134, depending on the PRE used. Sequential transference experiments showed that the behavior of the 4-member co-culture relies on the type of PRE provided for growth.

Conclusions: The results suggest that individual strain behavior changed when they grew in combinations of two, three, or four members, and those changes are determined first by the inherent characteristics of each strain and secondly by the environment.

Keywords: Arabidopsis; Bacterial growth; Burkholderiaceae; Co-culture; Root exudates.

Fig. 1

Growth curves of the four Burkholderiaceae strains on Arabidopsis thaliana root exudates. Growth curves were obtained after 120 h in culture tests carried out with A. thaliana root exudates (PRE) collected on day 14, or 21, with plants grown on standard (PRE) or N-limiting (N-PRE) conditions: (A) 14d.PRE; (B) 21d.PRE; (C) 14d.N-PRE, and (D) 21d.N-PRE. Each growth curve corresponds to the average of three replicates of cultures inoculated with each individual strain alone, or the 4-member combination of Paraburkholderia phytofirmans PsJN, Cupriavidus pinatubonensis JMP134, C. metallidurans CH34, and C. taiwanensis LMG19424. Standard deviations were lower than 5–10% and are not shown for clarity

Fig. 2

First generation cluster discrimination. Graphic representation of cluster determination through K-means. Each panel (A) 14d.PRE; (B) 21d.PRE; (C) 14d.N-PRE, and (D) 21d.N-PRE, represent the output of the K-means clustering algorithm. On each panel, 20 growth curves were analyzed to obtain the clusters: eight replicates of the initial 4-member combination (Consortium_1 to Consortium_8) and three replicates for each individual culture: PsJN_1 to PsJN_3 for Paraburkholderia phytofirmans PsJN; JMP134_1 to JMP134_3 for Cupriavidus pinatubonensis JMP134; CH34_1 to CH34_3 for C. metallidurans CH34, and LMG19424_1 to LMG19424_3 for C. taiwanensis LMG19424. Clusters that contain the 4-member combination are colored light blue. A larger symbol is presented in each cluster centroid

Fig. 3

Percentage of variation in viable cell numbers of Burkholderiaceae strains growing on Arabidopsis root exudates when single cultures are compared with co-cultures combinations. Percentages of variation in viable cells were calculated after comparison of single cultures values with those for pairs, trios, and quartet combinations of Paraburkholderia phytofirmans PsJN, Cupriavidus pinatubonensis JMP134, C. metallidurans CH34, and C. taiwanensis LMG19424 cultures on A. thaliana root exudates (PRE) collected at day 14, or 21, with plants exposed to standard or N-limiting conditions (14d.PRE, 21d.PRE and 21d.N-PRE). The color code bar corresponds to decreases (red) or increases (blue)

Fig. 4

Growth curves of the 4-members Burkholderiaceae co-culture on Arabidopsis thaliana root exudates for seven generations: Growth curves obtained after 72 h in culture tests carried out with A. thaliana root exudates (PRE) collected at day 14, or 21, with plants grown on standard or N-limiting conditions: (A) 14d.PRE; (B) 21d.PRE; (C) 14d.N-PRE, and (D) 21d.N-PRE. Each growth curve corresponds to the average of eight replicates of the 4-member co-culture of Paraburkholderia phytofirmans PsJN, Cupriavidus pinatubonensis JMP134, C. metallidurans CH34, and C. taiwanensis LMG19424. Standard deviations were lower than 5 or 10% and are not shown for clarity

Fig. 5

Experimental design. Procedure to obtain Arabidopsis thaliana root exudate pools (A), used in the sequential transference experiment (B). Plant root exudates were collected from plants growing under standard or nitrogen limitation conditions after 14 or 21 days; 14d.PRE and 21d.PRE, and 14d.N-PRE and 21d.N-PRE, respectively. Eight replicates of the individual or combined inocula of Paraburkholderia phytofirmans PsJN, Cupriavidus pinatubonensis JMP134, C. metallidurans CH34, and C. taiwanensis LMG19424 were grown for 120, or 72 h. Growth curves, viable cell counting, and bacterial abundances were then determined. Three of the eight replicates were selected to inoculate the next generation. This process was repeated until the seventh generation. For more details on the selection procedure, see Additional File 2