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.
Project description:The production and quality of Rehmannia glutinosa can be dramatically reduced by replant disease under consecutive monoculture. The root-associated microbiome, also known as the second genome of the plant, was investigated to understand its impact on plant health. Culture-dependent and culture-independent pyrosequencing analysis was applied to assess the shifts in soil bacterial communities in the rhizosphere and rhizoplane under consecutive monoculture. The results show that the root-associated microbiome (including rhizosphere and rhizoplane microbiomes) was significantly impacted by rhizocompartments and consecutive monoculture. Consecutive monoculture of R. glutinosa led to a significant decline in the relative abundance of the phyla Firmicutes and Actinobacteria in the rhizosphere and rhizoplane. Furthermore, the families Flavobacteriaceae, Sphingomonadaceae, and Xanthomonadaceae enriched while Pseudomonadaceae, Bacillaceae, and Micrococcaceae decreased under consecutive monoculture. At the genus level, Pseudomonas, Bacillus, and Arthrobacter were prevalent in the newly planted soil, which decreased in consecutive monocultured soils. Besides, culture-dependent analysis confirmed the widespread presence of Pseudomonas spp. and Bacillus spp. in newly planted soil and their strong antagonistic activities against fungal pathogens. In conclusion, R. glutinosa monoculture resulted in distinct root-associated microbiome variation with a reduction in the abundance of beneficial microbes, which might contribute to the declined soil suppressiveness to fungal pathogens in the monoculture regime.
Project description:BACKGROUND: Cell-to-cell communication (quorum sensing (QS)) co-ordinates bacterial behaviour at a population level. Consequently the behaviour of a natural multi-species community is likely to depend at least in part on co-existing QS and quorum quenching (QQ) activities. Here we sought to discover novel N-acylhomoserine lactone (AHL)-dependent QS and QQ strains by investigating a bacterial community associated with the rhizosphere of ginger (Zingiber officinale) growing in the Malaysian rainforest. RESULTS: By using a basal growth medium containing N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) as the sole source of carbon and nitrogen, the ginger rhizosphere associated bacteria were enriched for strains with AHL-degrading capabilities. Three isolates belonging to the genera Acinetobacter (GG2), Burkholderia (GG4) and Klebsiella (Se14) were identified and selected for further study. Strains GG2 and Se14 exhibited the broadest spectrum of AHL-degrading activities via lactonolysis while GG4 reduced 3-oxo-AHLs to the corresponding 3-hydroxy compounds. In GG2 and GG4, QQ was found to co-exist with AHL-dependent QS and GG2 was shown to inactivate both self-generated and exogenously supplied AHLs. GG2, GG4 and Se14 were each able to attenuate virulence factor production in both human and plant pathogens. CONCLUSIONS: Collectively our data show that ginger rhizosphere bacteria which make and degrade a wide range of AHLs are likely to play a collective role in determining the QS-dependent phenotype of a polymicrobial community.
Project description:Consecutive monoculture practice facilitates enrichment of rhizosphere pathogenic microorganisms and eventually leads to the emergence of replant disease. However, little is known about the interaction relationship among pathogens enriched in rhizosphere soils, Nucleotide binding-leucine-rich repeats (NB-LRR) receptors that specifically recognize pathogens in effector-triggered immunity (ETI) and physiological indicators under replant disease stress in Rehmannia glutinosa. In this study, a controlled experiment was performed using different kinds of soils from sites never planted R. glutinosa (NP), replanted R. glutinosa (TP) and mixed by different ration of TP soils (1/3TP and 2/3TP), respectively. As a result, different levels of TP significantly promoted the proliferation of Fusarium oxysporum f.sp. R. glutinosa (FO). Simultaneously, a comparison between FO numbers and NB-LRR expressions indicated that NB-LRRs were not consecutively responsive to the FO proliferation at transcriptional levels. Further analysis found that NB-LRRs responded to FO invasion with a typical phenomenon of "promotion in low concentration and suppression in high concentration", and 6 NB-LRRs were identified as candidates for responding R. glutinosa replant disease. Furthermore, four critical hormones of salicylic acid (SA), jasmonic acid (JA), ethylene (ET) and abscisic acid (ABA) had higher levels in 1/3TP, 2/3TP and TP than those in NP. Additionally, increasing extents of SA contents have significantly negative trends with FO changes, which implied that SA might be inhibited by FO in replanted R. glutinosa. Concomitantly, the physiological indexes reacted alters of cellular process regulated by NB-LRR were affected by complex replant disease stresses and exhibited strong fluctuations, leading to the death of R. glutinosa. These findings provide important insights and clues into further revealing the mechanism of R. glutinosa replant disease.
Project description:There is increasing evidence being accumulated regarding the importance of N-acyl homoserine lactones (AHL)-mediated quorum-sensing (QS) and quorum-quenching (QQ) processes in the marine environment, but in most cases, data has been obtained from specific microhabitats, and subsequently little is known regarding these activities in free-living marine bacteria. The QS and QQ activities among 605 bacterial isolates obtained at 90 and 2000 m depths in the Mediterranean Sea were analyzed. Additionally, putative QS and QQ sequences were searched in metagenomic data obtained at different depths (15-2000 m) at the same sampling site. The number of AHL producers was higher in the 90 m sample (37.66%) than in the 2000 m sample (4.01%). However, the presence of QQ enzymatic activity was 1.63-fold higher in the 2000 m sample. The analysis of putative QQ enzymes in the metagenomes supports the relevance of QQ processes in the deepest samples, found in cultivable bacteria. Despite the unavoidable biases in the cultivation methods and biosensor assays and the possible promiscuous activity of the QQ enzymes retrieved in the metagenomic analysis, the results indicate that AHL-related QS and QQ processes could be common activity in the marine environment.
Project description:Many Gram-negative plant pathogenic bacteria employ a N-acylhomoserine lactone (AHL)-based quorum sensing (QS) system to regulate their virulence traits. A sustainable biocontrol strategy has been developed using quorum quenching (QQ) bacteria to interfere with QS and protect plants from pathogens. Here, the prevalence and the diversity of QQ strains inhabiting tobacco leaf surfaces were explored. A total of 1177 leaf-associated isolates were screened for their ability to disrupt AHL-mediated QS, using the biosensor Chromobacterium violaceum CV026. One hundred and sixty-eight strains (14%) are capable of interfering with AHL activity. Among these, 106 strains (63%) of the culturable quenchers can enzymatically degrade AHL molecules, while the remaining strains might use other QS inhibitors to interrupt the chemical communication. Moreover, almost 79% of the QQ strains capable of inactivating AHLs enzymatically have lactonase activity. Further phylogenetic analysis based on 16S rDNA revealed that the leaf-associated QQ bacteria can be classified as Bacillus sp., Acinetobacter sp., Lysinibacillus sp., Serratia sp., Pseudomonas sp., and Myroides sp. The naturally occurring diversity of bacterial quenchers might provide opportunities to use them as effective biocontrol reagents for suppressing plant pathogen in situ.
Project description:Consecutive monoculture of Rehmannia glutinosa, highly valued in traditional Chinese medicine, leads to a severe decline in both quality and yield. Rhizosphere microbiome was reported to be closely associated with the soil health and plant performance. In this study, comparative metagenomics was applied to investigate the shifts in rhizosphere microbial structures and functional potentials under consecutive monoculture. The results showed R. glutinosa monoculture significantly decreased the relative abundances of Pseudomonadaceae and Burkholderiaceae, but significantly increased the relative abundances of Sphingomonadaceae and Streptomycetaceae. Moreover, the abundances of genera Pseudomonas, Azotobacter, Burkholderia, and Lysobacter, among others, were significantly lower in two-year monocultured soil than in one-year cultured soil. For potentially harmful/indicator microorganisms, the percentages of reads categorized to defense mechanisms (i.e., ATP-binding cassette (ABC) transporters, efflux transporter, antibiotic resistance) and biological metabolism (i.e., lipid transport and metabolism, secondary metabolites biosynthesis, transport and catabolism, nucleotide transport and metabolism, transcription) were significantly higher in two-year monocultured soil than in one-year cultured soil, but the opposite was true for potentially beneficial microorganisms, which might disrupt the equilibrium between beneficial and harmful microbes. Collectively, our results provide important insights into the shifts in genomic diversity and functional potentials of rhizosphere microbiome in response to R. glutinosa consecutive monoculture.
Project description:N-Acyl homoserine lactones (AHLs) are signaling molecules used in the quorum sensing (QS) of Gram-negative bacteria. Some bacteria interfere with the QS system using AHL-inactivating enzymes, commonly known as quorum-quenching (QQ) enzymes. We have recently isolated a new QQ bacterium showing high resistance to multiple ?-lactam antibiotics, and its QQ enzyme (MacQ) confers ?-lactam antibiotic resistance and exhibits QQ activities. This observation suggests the possibility of isolating novel QQ bacteria from ?-lactam antibiotic-resistant bacteria. In this direction, we attempted to isolate penicillin G (PENG)-resistant bacteria from penicillin-contaminated river sediments and activated sludge treating penicillin-containing wastewater and characterize their QQ activities. Of 19 PENG-resistant isolates, six isolates showed high QQ activity toward a broad range of AHLs, including AHLs with 3-oxo substituents. Five of the six AHL-degraders showed AHL-acylase activity and hydrolyzed the amide bond of AHLs, whereas the remaining one strain did not show AHL-acylase activity, suggesting that this isolate may likely possess alternative degradation mechanism such as AHL-lactonase activity hydrolyzing the lactone ring of AHLs. The 16S rRNA gene sequence analysis results categorized these six AHL-degrading isolates into at least five genera, namely, Sphingomonas (Alphaproteobacteria), Diaphorobacter (Betaproteobacteria), Acidovorax (Betaproteobacteria), Stenotrophomonas (Gammaproteobacteria), and Mycobacterium (Actinobacteria); of these, Mycobacterium sp. M1 has never been known as QQ bacteria. Moreover, multiple ?-lactam antibiotics showed high minimum inhibitory concentrations (MICs) when tested against all of isolates. These results strongly demonstrate that a wide variety of ?-lactam antibiotic-resistant bacteria possess QQ activities. Although the genetic and enzymatic elements are yet unclear, this study may infer the functional and evolutionary correlation between ?-lactam antibiotic resistance and QQ activities.
Project description:BACKGROUND: The consecutive monoculture for most of medicinal plants, such as Rehmannia glutinosa, results in a significant reduction in the yield and quality. There is an urgent need to study for the sustainable development of Chinese herbaceous medicine. METHODOLOGY/PRINCIPAL FINDINGS: Comparative metaproteomics of rhizosphere soil was developed and used to analyze the underlying mechanism of the consecutive monoculture problems of R. glutinosa. The 2D-gel patterns of protein spots for the soil samples showed a strong matrix dependency. Among the spots, 103 spots with high resolution and repeatability were randomly selected and successfully identified by MALDI TOF-TOF MS for a rhizosphere soil metaproteomic profile analysis. These proteins originating from plants and microorganisms play important roles in nutrient cycles and energy flow in rhizospheric soil ecosystem. They function in protein, nucleotide and secondary metabolisms, signal transduction and resistance. Comparative metaproteomics analysis revealed 33 differentially expressed protein spots in rhizosphere soil in response to increasing years of monoculture. Among them, plant proteins related to carbon and nitrogen metabolism and stress response, were mostly up-regulated except a down-regulated protein (glutathione S-transferase) involving detoxification. The phenylalanine ammonia-lyase was believed to participate in the phenylpropanoid metabolism as shown with a considerable increase in total phenolic acid content with increasing years of monoculture. Microbial proteins related to protein metabolism and cell wall biosynthesis, were up-regulated except a down-regulated protein (geranylgeranyl pyrophosphate synthase) functioning in diterpenoid synthesis. The results suggest that the consecutive monoculture of R. glutinosa changes the soil microbial ecology due to the exudates accumulation, as a result, the nutrient cycles are affected, leading to the retardation of plant growth and development. CONCLUSIONS/SIGNIFICANCE: Our results demonstrated the interactions among plant, soil and microflora in the proteomic level are crucial for the productivity and quality of R. glutinosa in consecutive monoculture system.
Project description:Production of medicinal tubers of Rehmannia glutinosa is severely hindered by replanting issues. However, a mechanistic understanding of the plant-soil factors associated with replant problems is currently limited. Thus, we aimed to identify the R. glutinosa root exudates, evaluate their potential phytotoxicity and profile the interactions between the plant and its associated rhizobiome. Stereomicroscopy and liquid chromatography coupled to a quadrupole/time of flight mass spectrometer were used to monitor and identify secreted metabolites, respectively. Seedling bioassays were used to evaluate the phytotoxicity of R. glutinosa root exudates. Two complimentary experiments were performed to investigate allelochemical fate in rhizosphere soil and profile the associated microbiota. Root specific microbes were further isolated from R. glutinosa rhizosphere. Impacts of isolated strains were evaluated by co-cultivation on plate and on seedlings in tissue culture, with a focus on their pathogenicity. Interactions between key R. glutinosa root exudates and isolated rhizobiomes were investigated to understand the potential for plant-soil feedbacks. Quantification and phytotoxic analysis of metabolites released from R. glutinosa indicated catalpol was the most abundant and bioactive metabolite in root exudates. Subsequent microbial profiling in soil containing accumulated and ecologically significant levels of catalpol identified several taxa (e.g., Agromyces, Lysobacter, Pseudomonas, Fusarium) that were specifically shifted. Isolation of R. glutinosa rhizobiomes obtained several root specific strains. A significant antagonistic effect between strain Rh7 (Pseudomonas aeruginosa) and two pathogenic strains Rf1 (Fusarium oxysporum) and Rf2 (Fusarium solani) was observed. Notably, the growth of strain Rh7 and catalpol concentration showed a hormesis-like effect. Field investigation further indicated catalpol was increasingly accumulated in the rhizosphere of replanted R. glutinosa, suggesting that interactions of biocontrol agents and pathogens are likely regulated by the presence of bioactive root exudates and in turn impact the rhizo-ecological process. In summary, this research successfully monitored the release of R. glutinosa root exudates, identified several abundant bioactive R. glutinosa secreted metabolites, profiled associated root specific microbes, and investigated the plant-soil feedbacks potentially regulated by catalpol and associated rhizobiomes. Our findings provide new perspectives toward an enhanced understanding R. glutinosa replant problems.
Project description:Under consecutive monoculture, the biomass and quality of Rehmannia glutinosa declines significantly. Consecutive monoculture of R. glutinosa in a four-year field trial led to significant growth inhibition. Most phenolic acids in root exudates had cumulative effects over time under sterile conditions, but these effects were not observed in the rhizosphere under monoculture conditions. It suggested soil microbes might be involved in the degradation and conversion of phenolic acids from the monocultured plants. T-RFLP and qPCR analysis demonstrated differences in both soil bacterial and fungal communities during monoculture. Prolonged monoculture significantly increased levels of Fusarium oxysporum, but decreased levels of Pseudomonas spp. Abundance of beneficial Pseudomonas spp. with antagonistic activity against F. oxysporum was lower in extended monoculture soils. Phenolic acid mixture at a ratio similar to that found in the rhizosphere could promote mycelial growth, sporulation, and toxin (3-Acetyldeoxynivalenol, 15-O-Acetyl-4-deoxynivalenol) production of pathogenic F. oxysporum while inhibiting growth of the beneficial Pseudomonas sp. W12. This study demonstrates that extended monoculture can alter the microbial community of the rhizosphere, leading to relatively fewer beneficial microorganisms and relatively more pathogenic and toxin-producing microorganisms, which is mediated by the root exudates.