Water-limiting conditions alter the structure and biofilm-forming ability of bacterial multispecies communities in the alfalfa rhizosphere

Abstract

Biofilms are microbial communities that adhere to biotic or abiotic surfaces and are enclosed in a protective matrix of extracellular compounds. An important advantage of the biofilm lifestyle for soil bacteria (rhizobacteria) is protection against water deprivation (desiccation or osmotic effect). The rhizosphere is a crucial microhabitat for ecological, interactive, and agricultural production processes. The composition and functions of bacterial biofilms in soil microniches are poorly understood. We studied multibacterial communities established as biofilm-like structures in the rhizosphere of Medicago sativa (alfalfa) exposed to 3 experimental conditions of water limitation. The whole biofilm-forming ability (WBFA) for rhizospheric communities exposed to desiccation was higher than that of communities exposed to saline or nonstressful conditions. A culture-dependent ribotyping analysis indicated that communities exposed to desiccation or saline conditions were more diverse than those under the nonstressful condition. 16S rRNA gene sequencing of selected strains showed that the rhizospheric communities consisted primarily of members of the Actinobacteria and α- and γ-Proteobacteria, regardless of the water-limiting condition. Our findings contribute to improved understanding of the effects of environmental stress factors on plant-bacteria interaction processes and have potential application to agricultural management practices.

Figure 1. Total cultivable bacterial counts in 3 types of alfalfa rhizospheric soils.

Counts of cultivable bacteria in rhizospheric soils exposed to nonstressful condition (CRS) and water-limiting conditions (DRS and SRS) are expressed as log₁₀ CFU per g dry rhizospheric soil. The values and error bars are mean and S.D. of 4 replicates per treatment. Differing letters indicate significant differences (P< 0.05) between counts.

Figure 2. Growth of alfalfa plants exposed to 3 experimental conditions.

Aerial development of alfalfa plants at the F2 stage (preflowering) under CRS, DRS, and SRS conditions.

Figure 3. Whole planktonic growth (A), WBFA (B), and relative BFA (C) of bacterial communities from 3 types of alfalfa rhizospheric soil.

The values are means of OD₆₂₀ (planktonic growth), OD₅₇₀ (biofilm formation quantified by staining with crystal violet), or their ratio (B/G) obtained for each plate (~95 strains) and averaged from 5 independent replicates for each treatment. The image at the bottom of Panel B shows the actual plates for each treatment. Differing letters indicate significant differences between treatments according to Fisher’s LSD test (P< 0.05).

Figure 4. BFA of bacterial subpopulations (HBFA and LBFA) isolated from 3 types of alfalfa rhizospheric soil.

BFA of 15 strains grouped as HBFA (A) and LBFA (B) subpopulations of CRS, DRS, and SRS communities as explained in the text. The bars indicate the mean value of OD₅₇₀ (biofilm formation quantified by staining with crystal violet) from 5 independent replicates for each treatment. Differing letters indicate significant differences between treatments according to Fisher’s LSD test (P< 0.05).

Figure 5. Distribution of ribotypes for HBFA (A) and LBFA (B) strains isolated from 3 types of alfalfa rhizospheric soil.

Each "slice" corresponds to a particular restriction profile obtained by digestion of amplified 16S rRNA gene (ribotype; R) with restriction endonuclease HaeIII. The number in parentheses is the number of strains that shared the ribotype for the particular treatment. Unique ribotypes are indicated by white. Shared ribotypes are indicated by light gray, dark gray, or black.

Figure 6. Dendrograms based on RFLP of 16S rRNA gene analysis using the UPGMA algorithm.

A: Dendrogram generated for the 3 types of alfalfa rhizospheric soil according to the ribotypes of the strains isolated. B: Dendrogram generated for the 3 types of soil in combination with the BFA (LBFA vs. HBFA) of the bacterial subpopulations, according to the ribotypes of the strains isolated.

Figure 7. Relationships among BFA-related traits, ribotypes, phylogenetic affiliations, and experimental treatments.

The graph was obtained from PCA using the InfoStat software program, version 2.0. Diamonds indicate combinations of ribotypes and affiliations with treatments. R: ribotype. Act: Actinobacteria. Prot: Proteobacteria. C: CRS. D: DRS. S: SRS. H: HBFA. L: LBFA. Circles indicate biological variables: BFA, biofilm-forming ability; MOT, motility; AGG, autoaggregation; AHL, production of QS signal; EPS, EPS production. The angles formed between the straight lines indicate the degree of correlation between variables (see text). PC1: Principal Component 1. PC2: Principal Component 2.