Linking Short-Chain N-Acyl Homoserine Lactone-Mediated Quorum Sensing and Replant Disease: A Case Study of Rehmannia glutinosa

Abstract

Rehmannia glutinosa, a perennial medicinal plant, suffers from severe replant disease under consecutive monoculture. The rhizosphere microbiome is vital for soil suppressiveness to diseases and for plant health. Moreover, N-acyl homoserine lactone (AHL)-mediated quorum sensing (QS) regulates diverse behavior in rhizosphere-inhabiting and plant pathogenic bacteria. The dynamics of short-chain AHL-mediated QS bacteria driven by consecutive monoculture and its relationships with R. glutinosa replant disease were explored in this study. The screening of QS bacteria showed that 65 out of 200 strains (32.5%) randomly selected from newly planted soil of R. glutinosa were detected as QS bacteria, mainly consisting of Pseudomonas spp. (55.4%). By contrast, 34 out of 200 (17%) strains from the diseased replant soil were detected as QS bacteria, mainly consisting of Enterobacteriaceae (73.5%). Functional analysis showed most of the QS bacteria belonging to the Pseudomonas genus showed strong antagonistic activities against Fusarium oxysporum or Aspergillus flavus, two main causal agents of R. glutinosa root rot disease. However, the QS strains dominant in the replant soil caused severe wilt disease in the tissue culture seedlings of R. glutinosa. Microbial growth assays demonstrated a concentration-dependent inhibitory effect on the growth of beneficial QS bacteria (i.e., Pseudomonas brassicacearum) by a phenolic acid mixture identified in the root exudates of R. glutinosa, but the opposite was true for harmful QS bacteria (i.e., Enterobacter spp.). Furthermore, it was found that the population of quorum quenching (QQ) bacteria that could disrupt the beneficial P. brassicacearum SZ50 QS system was significantly higher in the replant soil than in the newly planted soil. Most of these QQ bacteria in the replant soil were detected as Acinetobacter spp. The growth of specific QQ bacteria could be promoted by a phenolic acid mixture at a ratio similar to that found in the R. glutinosa rhizosphere. Moreover, these quorum-quenching bacteria showed strong pathogenicity toward the tissue culture seedlings of R. glutinosa. In conclusion, consecutive monoculture of R. glutinosa contributed to the imbalance between beneficial and harmful short-chain AHL-mediated QS bacteria in the rhizosphere, which was mediated not only by specific root exudates but also by the QQ bacterial community.

Keywords: Rehmannia glutinosa; quorum quenching; quorum sensing; replant disease; root exudate.

FIGURE 1

Screening of short-chain AHL-mediated QS bacteria by biosensor strain CV026 (A–C) and the dynamic changes of QS bacteria in the R. glutinosa rhizosphere under consecutive monoculture (D). (A) The biosensor strain CV026 did not produce the purple pigmentation when co-cultured with a known non-QS bacterium. (B) CV026 produced the purple pigmentation when inoculated with C6-HSL standard, as indicated by a dashed box. (C) The CV026 colony developed a purple color when co-cultured with a QS bacterium randomly isolated from the R. glutinosa rhizosphere.

FIGURE 2

Quantitative PCR of Pseudomonas sp. in the newly planted (NP) soil and the diseased (BT) soil of R. glutinosa. ** indicates a statistically significant difference (P < 0.01, LSD test). Data are presented as means ± standard errors.

FIGURE 3

Antagonistic activity assessment of QS bacteria (A) and phylogenetic analysis of QS antagonists (B). (A) Strains SZ107 and SZ109, rather than SZ110 and SZ112, had antagonistic activities against A. flavus (AF). The prefixes “SZ” and “BT” indicate the QS bacteria isolated from the newly planted (NP) soil and the diseased (BT) soil, respectively.

FIGURE 4

Effects of phenolic acid mixture on the growth of specific QS bacteria isolated from the newly planted (NP) soil and the diseased (BT) soil of R. glutinosa. The prefixes “SZ” and “BT” indicate the QS bacteria isolated from the NP soil and the BT soil, respectively. Strains S8, S9, SZ50, and SZ63 belong to the Pseudomonas genus. Strain BT3 belongs to the Pantoea genus, strains BT22 and BT23 belong to the Enterobacter genus and strain BT56 belongs to the Klebsiella genus. Data are presented using means ± standard errors. Different letters indicate different levels of significance (P < 0.05, LSD, n = 3).

FIGURE 5

The species composition and population dynamics of rhizosphere bacteria with QQ activities against Pseudomonas SZ50 under consecutive R. glutinosa monoculture. NP, CC, and BT represent the newly planted soil, the 2-year consecutively cropped soil and the diseased soil, respectively.