Complete genome sequence of Mucilaginibacter gossypii P3, a heavy metal(loid)-resistant bacterium, isolated from a gold and copper mine.

Mucilaginibacter gossypii P3, which was isolated from the sub-surface soil of the Zijin Gold and Copper Mine, displayed extremely high resistance to multiple heavy metal(loid)s and contained two novel ars operons. Complete genome sequencing of P3 yielded a single, closed genome of 7,187,928 bp, with GC content of 42.79%.

Key molecular events involved in root exudates-mediated replanted disease of Rehmannia glutinosa.

The perennial herbaceous plant, Rehmannia glutinosa Libosch, is one of traditional Chinese medicines with a long history of cultivation. However, replanted disease severely affects its yield and quality in production. In this study, a specific culture device was designed to accurately isolate the root exudates of R. glutinosa. In addition, the formation mechanism of replanted diseases mediated by root exudates was deeply studied in R. glutinosa. The results indicated that root exudates have obvious allelopathic activity, furthermore, metagenomics analysis found that the exudates were found to significantly induce the proliferation of harmful pathogenic fungal and the reduction of probiotics in rhizosphere of R. glutinosa. Further analysis found that, 8,758 genes were differentially expressed in root exudate-treated R. glutinosa plants. These genes mainly involved in critical cellular processes including immune response, hormone metabolism, signaling transduction and cell membrane transport. Of which, numerous genes were found to involve in immune response, such as PR (Pathogenesis-related protein), were highly expressed in root exudate-treated plants. Transiently overexpression experiments found that a PR1 could enhance the resistance of R. glutinosa to root exudates treatment. These results indicated that the interaction between root exudates and microbes altered the expression pattern of the genes related to immune pathway and signaling transduction mediated by it. These disordered genes finally severely affected the growth and development of R. glutinosa, and eventually formed the replanted disease. This study provides a novel approach to collect root exudates and a new data basis for revealing the molecular events occurring in replanted plants.

Rehmannia glutinosa Replant Issues: Root Exudate-Rhizobiome Interactions Clearly Influence Replant Success.

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.

NB-LRRs Not Responding Consecutively to Fusarium oxysporum Proliferation Caused Replant Disease Formation of Rehmannia glutinosa.

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.

Alleviatory effect of spent Pleurotus eryngii Quel substrate on replant problem of Rehmannia glutinosa Libosch.

Rehmannia glutinosa Libosch. is a medicinal plant cultivated at a commercial scale in China. However, replanting problems result in a severe decline in both the biomass and quality of its roots, which are of greatest medicinal value. This study attempted to remediate the replant soil using spent Pleurotus eryngii Quel substrate for alleviating this issue, and to investigate the underlying mechanisms. Results showed that R. glutinosa grew successfully in fresh soil and remedial replant soil, while no roots were harvested in the unremedied replant soil. Overall, the nutritional status in the remedial soil was higher than that of the unremedied and fresh soil, while the concentration of allelopathic phenolic acids was lower. When planted in unremedied soil, the growth of five plant pathogens was induced and one beneficial fungus was suppressed. When planted in remedied soil, four out of the five pathogens were suppressed, while two beneficial fungi were identified in the remedial soil. This study suggests that the spent P. eryngii substrate significantly alleviates the replant problem of R. glutinosa, and that the alleviatory function reflects a synergetic effect, including the supplementation of soil nutrition, the degradation of allelochemicals, and the remediation of unbalanced microbial community.

[Research progress of relationship between continuous cropping obstacles and microRNAs in Rehmannia glutinosa].

The efficacy of Rehmannia glutinosa which as a large quantity of traditional Chinese medicine is significant. However, the land must be given up after one season of R. glutinosa cultivation or replanted after a period of 8-10 years because of the severe continuous cropping obstacles. MicroRNAs is a class of endogenous non-coding small RNAs, which participate in regulation of physiological activities by target mRNA cleavage or translational repression in plants. In recent years,studies on the role of miRNAs in plants have made significant progresses,especially in medicinal plants．MiRNAs from some different medicinal plant species have been identified with regulatory effects．When plants are exposed to environmental stress, miRNAs act on stress-related genes and initiate stress-resistance mechanisms in the body against adverse factors. R. glutinosa is also a kind of environmental stress. It is conducive to deciphering the molecular mechanism of continuous cropping obstacles for us by researching miRNAs. This article reviews the production of miRNAs, mechanism, research approaches and characteristics of resisting the environmental stresses in plants, the development trends and future prospect of R. glutinosa miRNAs research.

[Advances on molecular mechanisms of Rehmannia glutinosa consecutive monoculture problem formation in multi-omics era].

Although consecutive monoculture problems have been studied for many years, no effective treatments are currently available. The complexity of systems triggered the formation of consecutive monoculture problems was one major cause. This paper elaborated the physiological and ecological mechanisms of consecutive monoculture problem formation based on the interaction relationship among multiple factors presented in the rhizosphere soil of consecutive monoculture plants. At same time, in this paper the multiple interactions among cultivated medicinal plants, autotoxic allelochemicals and rhizosphere microbial were proposed to be most important causes that derived the formation of consecutive monoculture problem. The paper also highlighted the advantage of 'omics' technologies integrating plant functional genomics and metabolomics as well as microbial macro-omics in understanding the multiple factor interaction under a particular ecological environment. Additionally, taking R. glutinosa as an example, the paper reviewed the molecular mechanism for the formation of R. glutinosa consecutive monoculture problem from the perspective of the accumulation of allelopathic autotoxins, the rhizosphere microecology catastrophe and theresponding of consecutive monoculture plants. Simultaneously, the roles of mutilple 'omics' technologies in comprehending these formation mechanism were described in detail. This paper provides finally a new insight to solve systematically the mechanism of consecutive monoculture problem formation on molecular level.

Differential proteomic analysis of replanted Rehmannia glutinosa roots by iTRAQ reveals molecular mechanisms for formation of replant disease.

BACKGROUND: The normal growth of Rehmannia glutinosa, a widely used medicinal plant in China, is severely disturbed by replant disease. The formation of replant disease commonly involves interactions among plants, allelochemicals and microbes; however, these relationships remain largely unclear. As a result, no effective measures are currently available to treat replant disease. RESULTS: In this study, an integrated R. glutinosa transcriptome was constructed, from which an R. glutinosa protein library was obtained. iTRAQ technology was then used to investigate changes in the proteins in replanted R. glutinosa roots, and the proteins that were expressed in response to replant disease were identified. An integrated R. glutinosa transcriptome from different developmental stages of replanted and normal-growth R. glutinosa produced 65,659 transcripts, which were accurately translated into 47,818 proteins. Using this resource, a set of 189 proteins was found to be significantly differentially expressed between normal-growth and replanted R. glutinosa. Of the proteins that were significantly upregulated in replanted R. glutinosa, most were related to metabolism, immune responses, ROS generation, programmed cell death, ER stress, and lignin synthesis. CONCLUSIONS: By integrating these key events and the results of previous studies on replant disease formation, a new picture of the damaging mechanisms that cause replant disease stress emerged. Replant disease altered the metabolic balance of R. glutinosa, activated immune defence systems, increased levels of ROS and antioxidant enzymes, and initiated the processes of cell death and senescence in replanted R. glutinosa. Additionally, lignin deposition in R. glutinosa roots that was caused by replanting significantly inhibited tuberous root formation. These key processes provide important insights into the underlying mechanisms leading to the formation of replant disease and also for the subsequent development of new control measures to improve production and quality of replanted plants.

Transcriptome analysis reveals metabolic alteration due to consecutive monoculture and abiotic stress stimuli in Rehamannia glutinosa Libosch.

KEY MESSAGE: We deeply investigated the mechanism underlying metabolic regulation in response to consecutive monoculture (replanting disease) and different abiotic stresses that unfolded the response mechanism to consecutive monoculture problem through RNA-seq analysis. The consecutive monoculture problem (CMP) resulted of complex environmental stresses mediated by multiple factors. Previous studies have noted that multiple stress factors in consecutive monoculture soils or plants severely limited the interpretation of the critical molecular mechanism, and made a predict that the specifically responding factor was autotoxic allelochemicals. To identify the specifically responding genes, we compared transcriptome changes in roots of Rehamannia glutinosa Libosch using consecutive monoculture, salt, drought, and ferulic acid as stress factors. Comparing with normal growth, 2502, 2672, 2485, and 1956 genes were differentially expressed in R. glutinosa under consecutive monoculture practice, salt, drought, and ferulic acid stress, respectively. In addition, 510 genes were specifically expressed under consecutive monoculture, which were not present under the other stress conditions. Integrating the biological and enrichment analyses of the differentially expressed genes, the result demonstrated that the plants could alter enzyme genes expression to reconstruct the complicated metabolic pathways, which used to tolerate the CMP and abiotic stresses. Furthermore, most of the affected pathway genes were closely related to secondary metabolic processes, and the influence of consecutive monoculture practice on the transcriptome genes expression profile was very similar to the profile under salt stress and then to the profile under drought stress. The outlined schematic diagram unfolded the putative signal regulation mechanism in response to the CMP. Genes that differentially up- or down-regulated under consecutive monoculture practice may play important roles in the CMP or replanting disease in R. glutinosa.

[Correlation of Allelopathy of Rehmannia glutinosa Root Exudates and Their Phenolic Acids Contents].

OBJECTIVE: To study the allelopathic potential of Rehmannia glutinosa root exudates in different growth stages and dynamic change of phenolic acids contents, in order to reveal the correlation between phenolic acids and allelbpathy effect of Rehmannia glutinosa. METHODS: Root exudates of Rehmannia glutinosa in different growth stages were obtained by a new instrument which was used to collect the root exudates of xerophytes. After that, bioassay was applied to estimate allelopathy effect of the root exudates. HPLC was used to determine the contents of five phenolic acids (coumaric acid, 4-hydroxybenzoic acid, vanillic acid, syringic acid and ferulic acid) which were reported to relate to allelopathy effect. Correlation of bioassay data and HPLC data were also analyzed. RESULTS: The germination rate of radish after soaking by root exudates of different growth stages of Rehmannia glutinosa was 97. 89%, 92. 38%, 89. 52%, 85. 71%, 85. 71%, 84. 76% and 83. 81%, respectively, which indicated a decline trend. And significant differences were shown from previous enlargement stage compared with the contrast. The bud length after soaking by root exudates was 5. 68, 5. 76, 5. 91, 5. 65, 5. 41, 5. 28 and 5. 11 cm, separately, which increased slightly before decreasing gradually. Previous enlargement stage was also the initial period when significant differences were shown. Five phenolic acids were detected in root exudates by HPLC, while the change of their contents and the allelopathy effect of root exudates did not perform a similar trend. Correlation analysis indicated the five phenolic acids did not have significant relevance (r = - 0. 666 - 0. 590) with germination rate and bud length of radish except the negative correlation (r = -0. 833, P <0. 05) of syringic acid and bud length. CONCLUSION: Significant allelopathy effect of Rehmannia glutinosa is performed from previous enlargement stage and enhanced with its growth. Syringic acid is a probable dominant allelochemical of Rehmannia glutinosa.

Analysis of integrated multiple 'omics' datasets reveals the mechanisms of initiation and determination in the formation of tuberous roots in Rehmannia glutinosa.

All tuberous roots in Rehmannia glutinosa originate from the expansion of fibrous roots (FRs), but not all FRs can successfully transform into tuberous roots. This study identified differentially expressed genes and proteins associated with the expansion of FRs, by comparing the tuberous root at expansion stages (initiated tuberous root, ITRs) and FRs at the seedling stage (initiated FRs, IFRs). The role of miRNAs in the expansion of FRs was also explored using the sRNA transcriptome and degradome to identify miRNAs and their target genes that were differentially expressed between ITRs and FRs at the mature stage (unexpanded FRs, UFRs, which are unable to expand into ITRs). A total of 6032 genes and 450 proteins were differentially expressed between ITRs and IFRs. Integrated analyses of these data revealed several genes and proteins involved in light signalling, hormone response, and signal transduction that might participate in the induction of tuberous root formation. Several genes related to cell division and cell wall metabolism were involved in initiating the expansion of IFRs. Of 135 miRNAs differentially expressed between ITRs and UFRs, there were 27 miRNAs whose targets were specifically identified in the degradome. Analysis of target genes showed that several miRNAs specifically expressed in UFRs were involved in the degradation of key genes required for the formation of tuberous roots. As far as could be ascertained, this is the first time that the miRNAs that control the transition of FRs to tuberous roots in R. glutinosa have been identified. This comprehensive analysis of 'omics' data sheds new light on the mechanisms involved in the regulation of tuberous roots formation.

[Cloning and expression analysis of the expansin gene RgEXPA10 in Rehmannia glutinosa].

Using cDNA from Rehmannia glutinosa leaf as template, a 972 bp fragment of expansin gene which containing a 762 bp ORF that encoded 253 amino acids, was cloned, named RgEXPA10, which GenBank accession number for this gene is KF011918. A 1 207 bp genomic sequence of RgEXPA10 was amplified by PCR with leaf DNA as template, sequencing analysis revealed that three exons and two introns in RgEXPA10 genomic sequence, and which GenBank accession number is KF011919. Molecular and bioinformatic analyses indicated that RgEXPA10 protein have DPBB_1 and Pollen_allerg_1 domain, also including a 26 aa nuclear localization signal and a 19 aa transmembrane region. Phylogenetic analysis revealed that RgEXPA10 showed the highest homology with AtEXPA8 among the 26 α-expansins in Arabidopsis thaliana. However, the RgEXPA10 indicated the highest homology with the expansin from Solanum lycopersicum among 22 plant species. Expression patterns using qRT-PCR analysis showed that RgEXPA10 mainly expressed in unfolded leaf, followed by the tuberous root at stage of expanding period, and rarely expressed in senescing leaf. And RgEXPA10 showed higher expression level in tuberous root at 60 and 90 days after emergence. The transcription level of RgEXPA10 significantly reduced under all the three stresses including continuous cropping conditions, salinity and waterlogging. This study will lay foundations for molecular function in development and regulation of different stresses for R. glutinosa.

Profile of natural redox mediators production of laccase-producing fungus Pleurotus ostreatus.

Polycyclic aromatic hydrocarbons (PAHs) are highly toxic organic pollutants which are abundant and environmentally widespread. Anthracene is a simple PAH that can be oxidized by laccases, copper-containing oxidase enzymes, produced by some plants, fungi, and bacteria. In this work, the extracellular culture fluid (CF) of laccase-producing fungus Pleurotus ostreatus was separated to crude laccase (CL) and aqueous ultrafiltrate (AU) fractions. The rate of anthracene oxidation by CF was 68.7 % while oxidation by CL was only 27.8 %. The addition of AU enhanced anthracene oxidation rate by CL to 60.4 %, indicating that the natural redox-mediators were present in the CF. The laccase-catalyzed anthracene oxidation rate increased with increased AU concentration, implying that oxidation rate is positively related to the concentration of natural mediators when laccase activity is constant. The AU from fungal culture containing bran or straw enhanced laccase-catalyzed anthracene oxidation; this enhancement increased further with prolonged fungus-cultivation, implying that both bran and straw induce the natural mediators. Our findings suggest increasing natural mediator levels may be an alternative strategy to improve the biodegradability of laccase-producing fungi.