Spatio Temporal Influence of Isoflavonoids on Bacterial Diversity in the Soybean Rhizosphere

Abstract

High bacterial density and diversity near plant roots has been attributed to rhizodeposit compounds that serve as both energy sources and signal molecules. However, it is unclear if and how specific rhizodeposit compounds influence bacterial diversity. We silenced the biosynthesis of isoflavonoids, a major component of soybean rhizodeposits, using RNA interference in hairy-root composite plants, and examined changes in rhizosphere bacteriome diversity. We used successive sonication to isolate soil fractions from different rhizosphere zones at two different time points and analyzed denaturing gradient gel electrophoresis profiles of 16S ribosomal RNA gene amplicons. Extensive diversity analysis of the resulting spatio temporal profiles of soybean bacterial communities indicated that, indeed, isoflavonoids significantly influenced soybean rhizosphere bacterial diversity. Our results also suggested a temporal gradient effect of rhizodeposit isoflavonoids on the rhizosphere. However, the hairy-root transformation process itself significantly altered rhizosphere bacterial diversity, necessitating appropriate additional controls. Gene silencing in hairy-root composite plants combined with successive sonication is a useful tool to determine the spatio temporal effect of specific rhizodeposit compounds on rhizosphere microbial communities.Pioneering microbiology studies by L. Hiltner in the early 1900s showed that the highest microbial density in soils occurs very close to plant roots (Hinsinger and Marschner 2006). For example, a four-to fivefold increase in CFU was observed in root-surface scrapings as compared with soil samples 0.5 cm away from the roots (Clark 1940). Such changes are attributed to the rich carbon energy sources provided by the plant. Indeed, plants release, on average, 10 to 15% (Jones et al. 2009) of their photosynthetic assimilates into the rhizosphere, a process called rhizodeposition (Dennis et al. 2010). These rhizodeposits originate from sloughed off root border and root border-like cells from root tips, active root exudation, and cell lysis. Rhizodeposits are composed of sugars, amino acids, organic acids, fatty acids, proteins, ions, secondary metabolites, mucilage, water, and miscellaneous carbon-containing compounds (Bais et al. 2006;Dennis et al. 2010).Significant evidence accumulated over the years indicates that the composition of root microbial communities is influenced, in large part, by the plant species and its developmental stage (Micallef et al. 2009;Mougel et al. 2006;Weisskopf et al. 2006). Indeed, an intricate coevolution of plants and rhizosphere microbial communities was suggested by the observation that resident plants or their root exudates are capable of maintaining the biomass and diversity of soil fungal communities to a much greater extent than nonresident or introduced plants ). This is supported by the observation that invasive weeds have the ability to significantly influence native rhizosphere microbial communities to exert their dominance in new environments (Inder...

Root nodule symbiosis in Lotus japonicus drives the establishment of distinctive rhizosphere, root, and nodule bacterial communities

“…A likely scenario for this enrichment is a positive feedback mechanism in which host-initiated signaling leads to enrichment of symbionts in the root and rhizosphere, a hypothesis that is supported by the markedly similar patterns observed in plants grown under nitrogen-supplemented conditions that impede nitrogen fixation. Legume root-derived flavonoids are candidate diffusible signaling molecules in such a feedback mechanism because their profile was shown to change during root-nodule symbiosis (59,60) and their broad impact on soil bacterial communities (61,62), especially on symbiotic rhizobia, has been documented (63,64).…”

Root isoflavonoids and hairy root transformation influence key bacterial taxa in the soybean rhizosphere

“…S1) and consistent silencing of IFS genes and significant reduction in root isoflavonoids were confirmed by qPCR and HPLC analyses respectively (White et al ., ). Here, we amplified and sequenced 16S variable regions V1–V3 and V3–V5 from (i) bulk soybean field soil (SFS; 2 replicates) without soybean roots, (ii) proximal soil (White et al ., ) from unaltered soybean roots (UNR; 3 replicates), (iii) proximal soil from vector control roots (VC; 5 replicates) and (iv) proximal soil from IFS‐RNAi roots (IFSi; 5 replicates). High quality sequences of 16S amplicons (V1–V3 and V3–V5) were processed through an analysis pipeline (Supporting Information Table S2 and Supporting Information Fig.…”

“…Our second objective was to determine the impact of the hairy root transformation procedure on the bacterial community structure by comparing the UNR and VC samples. As we had previously reported using DGGE (White et al ., ), the samples acquired from the VC roots differed largely from those from UNR roots (Fig. ; Compare UNR vs. VC).…”

“…Statistical analyses of OTU bacterial community structures of these samples revealed a conservative difference between the IFSi and VC samples. This limited difference was also depicted in the subsequent comparisons of bacteria family proportions and supported by the denaturing gradient gel electrophoresis analysis in our previous study (White et al ., ). Of the 194–206 families detected by the V1–V3 and V3–V5 libraries, only 8 were notably affected by reduced isoflavonoid levels (6 increased, 2 reduced).…”

“…Our results showed there was a significant difference in the rhizosphere bacterial community diversity of roots with normal isoflavonoid levels compared with roots with reduced isoflavonoid levels. Additionally, they appeared to have a temporal gradient effect on the rhizosphere, with the isoflavonoids exerting greater influence as more time passed (White et al ., ). Although the study showed the basic impact of isoflavonoids on the soybean bacterial community diversity it did not clarify how the community was affected, such as whether specific bacterial groups were suppressed or enhanced.…”