Intercropping with Achyranthes bidentata alleviates Rehmannia glutinosa consecutive monoculture problem by reestablishing rhizosphere microenvironment.

Background: The consecutive monoculture of Rehmannia glutinosa leads to a serious decrease in its production and quality. Previous studies have demonstrated that intercropping altered species diversity and rhizosphere microbial diversity. However, it remained unknown whether the impaired growth of monocultured plants could be restored by enhanced belowground interspecific interactions. Method: In the present research, a continuous cropping facilitator Achyranthes bidentata was intercropped with R. glutinosa under pot conditions, and three different types of root barrier treatments were set, including that complete belowground interaction (N), partial belowground interaction (S), and no belowground interspecies interaction (M), with the aims to investigate belowground interaction and the underlying mechanism of alleviated replanting disease of R. glutinosa by intercropping with A. bidentata. Results: The results showed that the land equivalent ratio (LER) of the two years was 1.17, and the system productivity index (SPI) increased by 16.92 % under S treatment, whereas no significant difference was found in N and M regimes. In the rhizosphere soil, intercropping systems had significantly increased the contents of sugars and malic acid in the soil of R. glutinosa, together with the content of organic matter and the invertase and urease activities. Meanwhile, intercropping increased the community diversity of fungi and bacteria, and the relative abundance of potential beneficial bacteria, such as Bacillus, Nitrospira, and Sphingomonas, despite the pathogenic Fusarium oxysporum was still the dominant genus in the rhizospheric soil of R. glutinosa under various treatments. The results of antagonism experiments and exogenous addition of specific bacteria showed that Bacillus spp. isolated from rhizosphere soil had a significant antagonistic effect on the pathogen of R. glutinosa. Conlusion: Taken together, our study indicated that the R. glutinosa//A. bidentata intercropping systems alleviate the consecutive monoculture problem of R. glutinosa by recruiting beneficial bacteria. The studies we have conducted have a positive effect on sustainable agricultural development.

Effect of Burkholderia ambifaria LK-P4 inoculation on the plant growth characteristics, metabolism, and pharmacological activity of Anoectochilus roxburghii.

Background: Plant growth-promoting bacteria (PGPB) represents a common biological fertilizer with remarkable effect in improving crop production and environmental friendliness. Methods: In the present work, we presented a detailed characterization of plant morphology and physiology, metabolism, and pharmacological activity of A. roxburghii between Burkholderia ambifaria LK-P4 inoculation and un-inoculation (CK) treatment by routine analytical techniques (include microscopy and enzymatic activity assays and so on) coupled with metabolomics approaches. Results: Morphological and physiological results showedthat the P4 bacteria could significantly increase plant stomatal density, freshweight, survival rate,and the content of total flavonoids in leaves but reducethe amount of free amino acid. Furthermore, metabolite data showed that fatty acids (linoleic acid, linolenic acid, stearic acid) and active substance (kyotorphin and piceatannol) were specifically up-regulated in P4 inoculation. It was also demonstrated that the differential metabolites were involved in citrate cycle, glyoxylate and dicarboxylate metabolism, and biosynthesis of unsaturated fatty acids pathway. In addition, pharmacological efficacy found that A. roxburghii under P4 inoculation can significantly decrease (p < 0.05) blood glucose levels and protect the organs of mice with similar effect of Glibenclamide tablets. Conlusion: Overall, it can be seen that the exogenous P4 bacteria can promote the growth and increase content of special metabolites in A. roxburghii. This study provided theoretical basis and supported for the high-yield and high-quality bionic cultivation of A. roxburghii.

Bio-fertilizer Amendment Alleviates the Replanting Disease under Consecutive Monoculture Regimes by Reshaping Leaf and Root Microbiome.

Replanting disease is a growing problem in intensive agricultural systems. Application of bio-fertilizer containing beneficial microbes contributes to disease suppression and is a promising strategy to control replanting disease. However, the effect of both replanting disease and bio-fertilizer amendment on the assembly of crop microbiota in leaves and roots and their relationships to crop yield and quality remains elusive. In these experiments, roots and leaves of Radix pseudostellariae were collected from different consecutive monoculture and bio-fertilizer amended fields, and the associated microbiota were characterized by bacterial 16S rRNA gene sequencing and quantitative PCR. Consecutive monoculture altered the bacterial community structure and composition and significantly increased the abundance of potential pathogenic Ralstonia and Fusarium oxysporum in leaves and roots. Furthermore, bio-fertilizer application alleviated replanting disease by decreasing the pathogen load, increasing the potential beneficial genera Pseudomonas, Streptomyces, Paenibacillus, and Bradyrhizobium. The proportion of positive correlations in the co-occurrence network of bio-fertilizer application was the highest, implying that bio-fertilizer potentially enhanced ecological commensalism or mutualism of the bacterial community across the two compartments. Structural equation models indicated that bio-fertilizer had a positive and indirect effect on both yield and quality by shaping the leaf microbiota and the root microbiota. Our findings highlight the role of leaf and root microbiota on replanting disease, showing that bio-fertilizer contributes to alleviating replanting disease by improving microbe-microbe interactions.

Revealing Microbiome Structure and Assembly Process in Three Rhizocompartments of Achyranthes bidentata Under Continuous Monoculture Regimes.

The complex composition and interaction of root-associated microbes are critical to plant health and performance. In this study, we presented a detailed characterization of three rhizocompartment (rhizosphere, rhizoplane, and root) microbiomes of Achyranthes bidentata under different years of consecutive monoculture by deep sequencing in order to determine keystone microorganisms via co-occurrence network analysis. The network analysis showed that multiple consecutive monoculture (MCM, represented 5Y and 10Y) soils generated some distinct beneficial bacterial taxa such as Bacillus, Fictibacillus, Bradyrhizobium, Shinella, and Herbaspirillum. For fungi, Mortierella substituted for Fusarium in occupying an important position in different rhizocompartments under A. bidentate monoculture. Quantitative PCR analysis confirmed a significant increase in Bacillus, Pseudomonas, and Burkholderia spp. The results of the inoculation assay showed that addition of beneficial bacteria Bacillus subtilis 74 and Bacillus halodurans 75 significantly increased the root length and fresh weight of A. bidentata. Furthermore, three types of phytosterones, as the main allochemicals, were identified both in the rhizosphere soil and in culture medium under sterile conditions by LC-MS/MS. When looking at in vitro interactions, it was found that phytosterones displayed a positive interaction with dominant beneficial species (Bacillus amyloliquefaciens 4 and B. halodurans 75) and had a negative effect on the presence of the pathogenic fungi Fusarium solani and Fusarium oxysporum. Overall, this study demonstrated that consecutive monoculture of A. bidentata can alter the bacterial and fungal community by secreting root exudates, leading to recruitment of beneficial microbes and replacement of plant-specific pathogenic fungi with plant beneficial fungi.

Antagonistic Activity of Trichoderma spp. Against Fusarium oxysporum in Rhizosphere of Radix pseudostellariae Triggers the Expression of Host Defense Genes and Improves Its Growth Under Long-Term Monoculture System.

Under consecutive monoculture, the abundance of pathogenic fungi, such as Fusarium oxysporum in the rhizosphere of Radix pseudostellariae, negatively affects the yield and quality of the plant. Therefore, it is pertinent to explore the role of antagonistic fungi for the management of fungal pathogens such as F. oxysporum. Our PCR-denatured gradient gel electrophoresis (DGGE) results revealed that the diversity of Trichoderma spp. was significantly declined due to extended monoculture. Similarly, quantitative PCR analysis showed a decline in Trichoderma spp., whereas a significant increase was observed in F. oxysporum. Furthermore, seven Trichoderma isolates from the R. pseudostellariae rhizosphere were identified and evaluated in vitro for their potentiality to antagonize F. oxysporum. The highest and lowest percentage of inhibition (PI) observed among these isolates were 47.91 and 16.67%, respectively. In in vivo assays, the R. pseudostellariae treated with four Trichoderma isolates, having PI > 30%, was used to evaluate the biocontrol efficiency against F. oxysporum in which T. harzianum ZC51 enhanced the growth of the plant without displaying any disease symptoms. Furthermore, the expression of eight defense-related genes of R. pseudostellariae in response to a combination of F. oxysporum and T. harzianum ZC51 treatment was checked, and most of these defense genes were found to be upregulated. In conclusion, this study reveals that the extended monoculture of R. pseudostellariae could alter the Trichoderma communities in the plant rhizosphere leading to relatively low level of antagonistic microorganisms. However, T. harzianum ZC51 could inhibit the pathogenic F. oxysporum and induce the expression of R. pseudostellariae defense genes. Hence, T. harzianum ZC51 improves the plant resistance and reduces the growth inhibitory effect of consecutive monoculture problem.

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

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.

Biochar mediates microbial communities and their metabolic characteristics under continuous monoculture.

Biochar amendment has been extensively used to improve plant performance and suppress disease in monoculture systems; however, few studies have focused on the underlying control mechanisms of replanting disease. In this study, we assessed the effects of biochar application on Radix pseudostellariae plant growth, rhizosphere soil microbial communities, and the physiological properties of microorganisms in a consecutive monoculture system. We found that biochar addition had little impact on the physiological parameters of tissue cultures of R. pseudostellaria but did significantly mediate microbial abundance in the rhizosphere soil of different consecutive monoculture years, leading to decreases in the abundance of pathogenic Fusarium oxysporum, Talaromyces helicus, and Kosakonia sacchari. Furthermore, biochar amendment had negative effects on the growth of beneficial bacteria, such as Burkholderia ambifaria, Pseudomonas chlororaphis, and Bacillus pumilus. Metabolomic analysis indicated that biochar significantly influenced the metabolic processes of F. oxysporum while inhibiting the mycelial growth and abating the virulence on plants. In summary, this study details the potential mechanisms responsible for the biochar-stimulated changes in the abundances and metabolism of rhizosphere bacteria and fungi, decreases in the contents of pathogens, and therefore improvements in the environmental conditions for plants growth. Further research is needed to evaluate the effects of biochar in long-term field trials.

Nitrogen Fertilizer Amendment Alter the Bacterial Community Structure in the Rhizosphere of Rice (Oryza sativa L.) and Improve Crop Yield.

Availability of nitrogen (N) in soil changes the composition and activities of microbial community, which is critical for the processing of soil organic matter and health of crop plants. Inappropriate application of N fertilizer can alter the rhizosphere microbial community and disturb the soil N homeostasis. The goal of this study was to assess the effect of different ratio of N fertilizer at various early to late growth stages of rice, while keeping the total N supply constant on rice growth performance, microbial community structure, and soil protein expression in rice rhizosphere. Two different N regimes were applied, i.e., traditional N application (NT) consists of three sessions including 60, 30 and 10% at pre-transplanting, tillering and panicle initiation stages, respectively, while efficient N application (NF) comprises of four sessions, i.e., 30, 30, 30, and 10%), where the fourth session was extended to anthesis stage. Soil metaproteomics combined with Terminal Restriction Fragment Length Polymorphism (T-RFLP) were used to determine the rhizosphere biological process. Under NF application, soil enzymes, nitrogen utilization efficiency and rice yield were significantly higher compared to NT application. T-RFLP and qPCR analysis revealed differences in rice rhizosphere bacterial diversity and structure. NF significantly decreased the specific microbes related to denitrification, but opposite result was observed for bacteria associated with nitrification. Furthermore, soil metaproteomics analysis showed that 88.28% of the soil proteins were derived from microbes, 5.74% from plants, and 6.25% from fauna. Specifically, most of the identified microbial proteins were involved in carbohydrate, amino acid and protein metabolisms. Our experiments revealed that NF positively regulates the functioning of the rhizosphere ecosystem and further enabled us to put new insight into microbial communities and soil protein expression in rice rhizosphere.

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Rehmannia glutinosa, a perennial herbaceous species, belongs to the family Scrophularia-ceae. As a staple medicinal material, its tuberous roots are highly valued in traditional Chinese medicine. However, R. glutinosa suffers from serious consecutive monoculture problems in production, which leads to a decline in both productivity and quality. Phyllosphere bacteria, the most abundant component of phyllosphere microorganisms, play crucial roles in plant growth and health. Characterization of phyllosphere bacteria could provide new insights into the mechanisms of consecutive monoculture problems and their control measures. Meanwhile, the varied taxa could be served as an important indicator of consecutive monoculture problems. The barcoded pyrosequencing of 16S rDNA genes combined with a culture-dependent approach was applied to characterize the shifts of bacterial community structure and diversity in the phyllosphere under consecutive monoculture of R. glutinosa. The results showed that consecutive monoculture clearly affected bacterial community structure in the phyllosphere. The phyllosphere bacterial communities of the two-year monocultured (TY) and the diseased plants (DP) were more similar, and different from the one-year monocultured (OY). The evenness, Shannon and Simpson diversity indices were significantly lower in TY and DP than in OY. Species annotation showed that bacterial community in R. glutinosa phyllosphere mainly consisted of Proteobacteria (91.2%), Firmicutes (5.1%) and Actinobacteria (3.7%). There was no significant difference in the number of detected bacterial taxa. However, Proteobacteria was significantly increased while Firmicutes and Actinobacteria were significantly decreased under consecutive monoculture. At the genus level, the relative abundances of genera Exiguobacterium, Bacillus and Arthrobacter, potentially beneficial microorganisms, were significantly higher in OY than that in TY and DP, but it was opposite for the genus Pseudomonas. The results from the culture-dependent approach and pathogenicity test showed that Pseudomonas plecoglossicida D9, widely isolated from the diseased leaves, was highly pathogenic to leaves. In conclusion, R. glutinosa monoculture resulted in distinct phyllosphere bacterial community variation with the accumulation of pathogen loads at the expense of beneficial microorganisms, which could contribute to the occurrence of leaf disease symptoms,and aggravate R. glutinosa replant disease in a monoculture regime.

Comparative Metagenomic Analysis of Rhizosphere Microbial Community Composition and Functional Potentials under Rehmannia glutinosa Consecutive Monoculture.

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.

Rhizosphere Fungal Community Dynamics Associated with Rehmannia glutinosa Replant Disease in a Consecutive Monoculture Regime.

Consecutive monoculture of Rehmannia glutinosa in the same field leads to a severe decline in both quality and yield of tuberous roots, the most useful part in traditional Chinese medicine. Fungi are an important and diverse group of microorganisms in the soil ecosystem and play crucial roles in soil health. In this study, high-throughput pyrosequencing of internal transcribed spacer 2 ribosomal DNA amplicons was applied to gain insight into how consecutive monoculture practice influence and stimulate R. glutinosa rhizosphere and bulk soil fungal communities. The results from nonmetric multidimensional scaling ordination and clustering analysis revealed distinctive differences between rhizosphere and bulk soil fungal communities. However, longer-term monocultured bulk soils were more similar to the rhizosphere soils in comparison with the shorter-term monocultured bulk soils. Moreover, consecutive monoculture caused a gradual shift in the composition and structure of the soil fungal community. The cultivation of this plant led to the appearance of some exclusive operational taxonomic units in rhizosphere or bulk soils that were assigned to the genera Fusarium, Rhizoctonia, and so on. Furthermore, the sum of the relative abundance of species of Fusarium, Cylindrocarpon, and Gibberella (belonging to the family Nectriaceae); Rhizoctonia, Thanatephorus, and Ceratobasidium (belonging to the family Ceratobasidiaceae); and Lectera and Plectosporium (belonging to the family Plectosphaerellaceae) was significantly higher in consecutively monocultured (CM) than in newly planted (NP) soil in both rhizosphere and bulk soils. In particular, Fusarium abundance was significantly higher in CM than in NP in the rhizosphere, and higher in rhizosphere soils than in bulk soils for each treatment. A pathogenicity test showed that both Fusarium strains isolated were pathogenic to R. glutinosa seedlings. In addition, the culture filtrate and mycotoxins produced by Fusarium oxysporum significantly repressed the growth of the antagonistic bacterium, Pseudomonas aeruginosa. In conclusion, consecutive monoculture of R. glutinosa restructured the fungal communities in both rhizosphere and bulk soils but bulk effects developed more slowly over time in comparison with rhizosphere effects. Furthermore, microbial interactions might lead to a reduction in the abundance of beneficial microbes.

Barcoded Pyrosequencing Reveals a Shift in the Bacterial Community in the Rhizosphere and Rhizoplane of Rehmannia glutinosa under Consecutive Monoculture.

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.

Assessment of the Diversity of Pseudomonas spp. and Fusarium spp. in Radix pseudostellariae Rhizosphere under Monoculture by Combining DGGE and Quantitative PCR.

Radix pseudostellariae is a perennial tonic medicinal plant, with high medicinal value. However, consecutive monoculture of this plant in the same field results in serious decrease in both yield and quality. In this study, a 3-year field experiment was performed to identify the inhibitory effect of growth caused by prolonged monoculture of R. pseudostellariae. DGGE analysis was used to explore the shifts in the structure and diversity of soil Fusarium and Pseudomonas communities along a 3-year gradient of monoculture. The results demonstrated that extended monoculture significantly boosted the diversity of Fusarium spp., but declined Pseudomonas spp. diversity. Quantitative PCR analysis showed a significant increase in Fusarium oxysporum, but a decline in Pseudomonas spp. Furthermore, abundance of antagonistic Pseudomonas spp. possessing antagonistic ability toward F. oxysporum significantly decreased in consecutively monocultured soils. Phenolic acid mixture at the same ratio as detected in soil could boost mycelial and sporular growth of pathogenic F. oxysporum while inhibit the growth of antagonistic Pseudomonas sp. CJ313. Moreover, plant bioassays showed that Pseudomonas sp. CJ313 had a good performance that protected R. pseudostellariae from infection by F. oxysporum. In conclusion, this study demonstrated that extended monoculture of R. pseudostellariae could alter the Fusarium and Pseudomonas communities in the plant rhizosphere, leading to relatively low level of antagonistic microorganisms, but with relatively high level of pathogenic microorganisms.

The role of organic acids on microbial deterioration in the Radix pseudostellariae rhizosphere under continuous monoculture regimes.

A three-year field monoculture trial of Radix pseudostellariae and complementary laboratory studies were conducted to further elucidate the underlying mechanism responsible for significant decreases in the biomass yield and quality of R. pseudostellariae under continuous monoculture regimes. HPLC analysis indicated that continuous monoculture soil was rich in organic acids, which had cumulative effects over time. Further analysis suggested that the application of a mixture of organic acids significantly promoted growth of pathogenic fungi, and increased the expression of chemotaxis-related gene (cheA) and biofilm formation of the specific pathogenic Kosakonia sacchari. However, opposite reactions were observed in the case of Bacillus megaterium and Bacillus pumilus. Concurrently, the present results revealed that the mixed organic acids stimulated the production of toxins, as well as H2O2 in the pathogenic fungi. Furthermore, the presence of organic acids reflecting environmental conditions under monocropping had negative effects on the expression of the biocontrol-related genes, which resulted in attenuated antagonistic activities of plant growth-promoting rhizobacteria (PGPR) to suppress mycelial growth of the pathogenic fungi. These results help to unveil the mechanisms associated with how accumulated organic acids differentially mediate deterioration of soil microbial composition and structure in monocropping system.

Insights into the Mechanism of Proliferation on the Special Microbes Mediated by Phenolic Acids in the Radix pseudostellariae Rhizosphere under Continuous Monoculture Regimes.

As potent allelochemicals, phenolic acids are believed to be associated with replanting disease and cause microflora shift and structural disorder in the rhizosphere soil of continuously monocultured Radix pseudostellariae. The transcriptome sequencing was used to reveal the mechanisms underlying the differential response of pathogenic bacterium Kosakonia sacchari and beneficial bacterium Bacillus pumilus on their interactions with phenolic acids, the main allelochemicals in root exudates of R. pseudostellariae in the monoculture system. The microbes were inoculated in the pots containing soil and the medicinal plant in this study. The results showed that the addition of beneficial B. pumilus to the 2-year planted soil significantly decreased the activity of soil urease, catalase, sucrase, and cellulase and increased the activity of chitinase compared with those in the 2nd-year monocropping rhizosphere soil without any treatment. However, opposite results were obtained when K. sacchari was added. Transcriptome analysis showed that vanillin enhanced glycolysis/gluconeogenesis, fatty acid biosynthesis, pentose phosphate, bacterial chemotaxis, flagellar assembly, and phosphotransferase system pathway in K. sacchari. However, protocatechuic acid, a metabolite produced by K. sacchari from vanillin, had negative effects on the citrate cycle and biosynthesis of novobiocin, phenylalanine, tyrosine, and tryptophan in B. pumilus. Concurrently, the protocatechuic acid decreased the biofilm formation of B. pumilus. These results unveiled the mechanisms how phenolic acids differentially mediate the shifts of microbial flora in rhizosphere soil, leading to the proliferation of pathogenic bacteria (i.e., K. sacchari) and the attenuation of beneficial bacteria (i.e., B. pumilus) under the monocropping system of R. pseudostellariae.

Insights into the Regulation of Rhizosphere Bacterial Communities by Application of Bio-organic Fertilizer in Pseudostellaria heterophylla Monoculture Regime.

The biomass and quality of Pseudostellariae heterophylla suffers a significant decline under monoculture. Since rhizosphere miobiome plays crucial roles in soil health, deep pyrosequencing combined with qPCR was applied to characterize the composition and structure of soil bacterial community under monoculture and different amendments. The results showed compared with the 1st-year planted (FP), 2nd-year monoculture of P. heterophylla (SP) led to a significant decline in yield and resulted in a significant increase in Fusarium oxysporum but a decline in Burkholderia spp. Bio-organic fertilizer (MT) formulated by combining antagonistic bacteria with organic matter could significantly promote the yield by regulating rhizosphere bacterial community. However, organic fertilizer (MO) without antagonistic bacteria could not suppress Fusarium wilt. Multivariate statistics analysis showed a distinct separation between the healthy samples (FP and MT) and the unhealthy samples (SP and MO), suggesting a strong relationship between soil microbial community and plant performance. Furthermore, we found the application of bio-organic fertilizer MT could significantly increase the bacterial community diversity and restructure microbial community with relatively fewer pathogenic F. oxysporum and more beneficial Burkholderia spp. In conclusion, the application of novel bio-organic fertilizer could effectively suppress Fusarium wilt by enriching the antagonistic bacteria and enhancing the bacterial diversity.

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.

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.