Linkun Wu

Comparative Metaproteomic Analysis on Consecutively Rehmannia glutinosa-Monocultured Rhizosphere Soil

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.

Characterization of metaproteomics in crop rhizospheric soil.

Soil rhizospheric metaproteomics is a powerful scientific tool to uncover the interactions between plants and microorganisms in the soil ecosystem. The present study established an extraction method suitable for different soils that could increase the extracted protein content. Close to 1000 separate spots with high reproducibility could be identified in the stained 2-DE gels. Among the spots, 189 spots representing 122 proteins on a 2-DE gel of rice soil samples were successfully identified by MALDI-TOF/TOF-MS. These proteins mainly originated from rice and microorganisms. They were involved in protein, energy, nucleotide, and secondary metabolisms, as well as signal transduction and resistance. Three characteristics of the crop rhizospheric metaproteomics seemed apparent: (1) approximately one-third of the protein spots could not be identified by MALDI-TOF/TOF/MS, (2) the conservative proteins from plants formed a feature distribution of crop rhizospheric metaproteome, and (3) there were very complex interactions between plants and microorganisms existing in a crop rhizospheric soil. Further functional analysis on the identified proteins unveiled various metabolic pathways and signal transductions involved in the soil biotic community. This study provides a paradigm for metaproteomic research on soil biology.

Plant-microbe rhizosphere interactions mediated by Rehmannia glutinosa root exudates under consecutive monoculture

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.

Metaproteomic analysis of ratoon sugarcane rhizospheric soil

BackgroundThe current study was undertaken to elucidate the mechanism of yield decline in ratoon sugarcane using soil metaproteomics combined with community level physiological profiles (CLPP) analysis.ResultsThe available stalk number, stalk diameter, single stalk weight and theoretical yield of ratoon cane (RS) were found to be significantly lower than those of plant cane (NS). The activities of several carbon, nitrogen and phosphorus processing enzymes, including invertase, peroxidase, urease and phosphomonoesterase were found to be significantly lower in RS soil than in NS soil. BIOLOG analysis indicated a significant decline in average well-color development (AWCD), Shannon’s diversity and evenness indices in RS soil as compared to NS soil. To profile the rhizospheric metaproteome, 109 soil protein spots with high resolution and repeatability were successfully identified. These proteins were found to be involved in carbohydrate/energy, amino acid, protein, nucleotide, auxin and secondary metabolisms, membrane transport, signal transduction and resistance, etc. Comparative metaproteomics analysis revealed that 38 proteins were differentially expressed in the RS soil as compared to the control soil or NS soil. Among these, most of the plant proteins related to carbohydrate and amino acid metabolism and stress response were up-regulated in RS soil. Furthermore, several microbial proteins related to membrane transport and signal transduction were up-regulated in RS soil. These proteins were speculated to function in root colonization by microbes.ConclusionsOur experiments revealed that sugarcane ratooning practice induced significant changes in the soil enzyme activities, the catabolic diversity of microbial community, and the expression level of soil proteins. They influenced the biochemical processes in the rhizosphere ecosystem and mediated the interactions between plants and soil microbes.

Interaction of Pseudostellaria heterophylla with Fusarium oxysporum f.sp. heterophylla mediated by its root exudates in a consecutive monoculture system

In this study, quantitative real-time PCR (qPCR) was used to determine the amount of Fusarium oxysporum, an important replant disease pathogen in Pseudostellaria heterophylla rhizospheric soil. Moreover, HPLC was used to identify phenolic acids in root exudates then it was further to explore the effects of the phenolic acid allelochemicals on the growth of F. oxysporum f.sp. heterophylla. The amount of F. oxysporum increased significantly in P. heterophylla rhizosphere soil under a consecutive replant system as monitored through qPCR analysis. Furthermore, the growth of F. oxysporum f.sp. heterophylla mycelium was enhanced by root exudates with a maximum increase of 23.8%. In addition, the number of spores increased to a maximum of 12.5-fold. Some phenolic acids promoted the growth of F. oxysporum f.sp. heterophylla mycelium and spore production. Our study revealed that phenolic acids in the root secretion of P. heterophylla increased long with its development, which was closely related to changes in rhizospheric microorganisms. The population of pathogenic microorganisms such as F. oxysporum in the rhizosphere soil of P. heterophylla also sharply increased. Our results on plant-microbe communication will help to better clarify the cause of problems associated with P. heterophylla under consecutive monoculture treatment.

Effects of consecutive monoculture of Pseudostellaria heterophylla on soil fungal community as determined by pyrosequencing

Under consecutive monoculture, the biomass and quality of Pseudostellaria heterophylla declines significantly. In this study, a three-year field experiment was conducted to identify typical growth inhibition effects caused by extended monoculturing of P. heterophylla. Deep pyrosequencing was used to examine changes in the structure and composition of soil fungal community along a three-year gradient of monoculture. The results revealed a distinct separation between the newly planted plot and the two-year, three-year monocultured plots. The Shannon and Simpson diversity indices were significantly higher in the two-year and three-year monoculture soils than in the newly planted soil. Consecutive monoculture of this plant led to a significant increase in relative abundance of Fusarium, Trichocladium and Myrothecium and Simplicillium, etc., but a significant decrease in the relative abundance of Penicillium. Quantitative PCR analysis confirmed a significant increase in Fusarium oxysporum, an agent known to cause wilt and rot disease of P. heterophylla. Furthermore, phenolic acid mixture at a ratio similar to that found in the rhizosphere could promote mycelial growth of pathogenic F. oxysporum. Overall, this study demonstrated that consecutive monoculture of P. heterophylla can alter the fungal community in the rhizosphere, including enrichment of host-specific pathogenic fungi at the expense of plant-beneficial fungi.

Mixed Phenolic Acids Mediated Proliferation of Pathogens Talaromyces helicus and Kosakonia sacchari in Continuously Monocultured Radix pseudostellariae Rhizosphere Soil

Radix pseudostellariae L. is a common and popular Chinese medication. However, continuous monoculture has increased its susceptibility to severe diseases. We identified two pathogenic microorganisms, Talaromyces helicus M. (KU355274) and Kosakonia sacchari W. (KU324465), and their antagonistic bacterium, Bacillus pumilus Z. in rhizosphere soil of continuously monocultured R. pseudostellariae. Nine types of phenolic acids were identified both in the rhizosphere soil and in culture medium under sterile conditions. A syringic acid and phenolic acid mixture significantly promoted the growth of T. helicus and K. sacchari. T. helicus could utilize eight types of phenolic acids, whereas K. sacchari could only use four phenolic acids. K. sacchari produced protocatechuic acid when consuming vanillin. Protocatechuic acid negatively affected the growth of B. pumilus. The 3A-DON toxin produced by T. helicus promoted the growth of K. sacchari and inhibited growth of B. pumilus at low concentrations. These data help explain why phenolic exudates mediate a microflora shift and structure disorder in the rhizosphere soil of continuously monocultured R. pseudostellariae and lead to increased replanting disease incidence.

Soil Microbial Community Structure and Metabolic Activity of Pinus elliottii Plantations across Different Stand Ages in a Subtropical Area

Soil microbes play an essential role in the forest ecosystem as an active component. This study examined the hypothesis that soil microbial community structure and metabolic activity would vary with the increasing stand ages in long-term pure plantations of Pinus elliottii. The phospholipid fatty acids (PLFA) combined with community level physiological profiles (CLPP) method was used to assess these characteristics in the rhizospheric soils of P. elliottii. We found that the soil microbial communities were significantly different among different stand ages of P. elliottii plantations. The PLFA analysis indicated that the bacterial biomass was higher than the actinomycic and fungal biomass in all stand ages. However, the bacterial biomass decreased with the increasing stand ages, while the fungal biomass increased. The four maximum biomarker concentrations in rhizospheric soils of P. elliottii for all stand ages were 18:1ω9c, 16:1ω7c, 18:3ω6c (6,9,12) and cy19:0, representing measures of fungal and gram negative bacterial biomass. In addition, CLPP analysis revealed that the utilization rate of amino acids, polymers, phenolic acids, and carbohydrates of soil microbial community gradually decreased with increasing stand ages, though this pattern was not observed for carboxylic acids and amines. Microbial community diversity, as determined by the Simpson index, Shannon-Wiener index, Richness index and McIntosh index, significantly decreased as stand age increased. Overall, both the PLFA and CLPP illustrated that the long-term pure plantation pattern exacerbated the microecological imbalance previously described in the rhizospheric soils of P. elliottii, and markedly decreased the soil microbial community diversity and metabolic activity. Based on the correlation analysis, we concluded that the soil nutrient and C/N ratio most significantly contributed to the variation of soil microbial community structure and metabolic activity in different stand ages of P. elliottii plantations.

Terminal Restriction Fragment Length Polymorphism Analysis of Soil Bacterial Communities under Different Vegetation Types in Subtropical Area

Soil microbes are active players in energy flow and material exchange of the forest ecosystems, but the research on the relationship between the microbial diversity and the vegetation types is less conducted, especially in the subtropical area of China. In this present study, the rhizosphere soils of evergreen broad-leaf forest (EBF), coniferous forest (CF), subalpine dwarf forest (SDF) and alpine meadow (AM) were chosen as test sites. Terminal-restriction fragment length polymorphisms (T-RFLP) analysis was used to detect the composition and diversity of soil bacterial communities under different vegetation types in the National Natural Reserve of Wuyi Mountains. Our results revealed distinct differences in soil microbial composition under different vegetation types. Total 73 microbes were identified in soil samples of the four vegetation types, and 56, 49, 46 and 36 clones were obtained from the soils of EBF, CF, SDF and AM, respectively, and subsequently sequenced. The Actinobacteria, Fusobacterium, Bacteroidetes and Proteobacteria were the most predominant in all soil samples. The order of Shannon-Wiener index (H) of all soil samples was in the order of EBF>CF>SDF>AM, whereas bacterial species richness as estimated by four restriction enzymes indicated no significant difference. Principal component analysis (PCA) revealed that the soil bacterial communities’ structures of EBF, CF, SDF and AM were clearly separated along the first and second principal components, which explained 62.17% and 31.58% of the total variance, respectively. The soil physical-chemical properties such as total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP) and total potassium (TK) were positively correlated with the diversity of bacterial communities.

Positive allelopathic stimulation and underlying molecular mechanism of achyranthe under continuous monoculture

Three experiments were conducted to study the allelopathic stimulation and its underlying molecular mechanism of achyranthes medicinal plants in continuously monoculture system. The stimulators in the rhizospheric soil of continuously monocultured achyranthes plants were extracted by water and organic solvents. Results of the bioassay showed that the rhizospheric soil extracts had a significant promotive effect on the growth of achyranthes in continuous monoculture system, implying that the extracts, especially the water extracts might contain plant activators to stimulate the growth of the medicinal plants. Subtractive hybridization suppression (SSH) was used to investigate gene expression profiles of achyranthes in response to the extract treatments. Ten up-regulated genes from SSH-cDNA library were sequenced and assigned. Results indicated that flavonoids and phytosterol might play an important role in the positively allelopathic stimulation on achyranthes plants in continuous monoculture system. Comparative proteomics were employed to further unveil the molecular mechanism of allelopathic stimulation induced by the extracts. Compared with protein expression profile in control, 25 differentially expressed proteins and their functions were detected and identified in the treated plants. The results suggested that the extracts from continuously monocultured rhizospheric soils under Chinese medicinal achyranthes activated the genes encoding the key enzymes involved in terpenes and flavonoids synthesis, which in turn led to increased de novo synthesis of the stimulators, and hence promoted growth of achyranthes in consecutively monoculture system.