Comprehensive genomic analysis of Burkholderia arboris PN-1 reveals its biocontrol potential against Fusarium solani-induced root rot in Panax notoginseng.

Panax notoginseng (Burkill) F.H. Chen, a valuable traditional Chinese medicine, faces significant yield and quality challenges stemming from root rot primarily caused by Fusarium solani. Burkholderia arboris PN-1, isolated from the rhizosphere soil of P. notoginseng, demonstrated a remarkable ability to inhibit the growth of F. solani. This study integrates phenotypic, phylogenetic, and genomic analyses to enhance our understanding of the biocontrol mechanisms employed by B. arboris PN-1. Phenotype analysis reveals that B. arboris PN-1 effectively suppresses P. notoginseng root rot both in vitro and in vivo. The genome of B. arboris PN-1 comprises three circular chromosomes (contig 1: 3,651,544 bp, contig 2: 1,355,460 bp, and contig 3: 3,471,056 bp), with a 66.81% GC content, housing 7,550 protein-coding genes. Notably, no plasmids were detected. Phylogenetic analysis places PN-1 in close relation to B. arboris AU14372, B. arboris LMG24066, and B. arboris MEC_B345. Average nucleotide identity (ANI) values confirm the PN-1 classification as B. arboris. Comparative analysis with seven other B. arboris strains identified 4,628 core genes in B. arboris PN-1. The pan-genome of B. arboris appears open but may approach closure. Whole-genome sequencing revealed 265 carbohydrate-active enzymes and identified 9 gene clusters encoding secondary metabolites. This comprehensive investigation enhances our understanding of B. arboris genomes, paving the way for their potential as effective biocontrol agents against fungal plant pathogens in the future.

Core rhizosphere microbiome of Panax notoginseng and its associations with belowground biomass and saponin contents.

The core rhizosphere microbiome is critical for plant fitness. However, its contribution to the belowground biomass and saponin contents of Panax notoginseng remains unclear. High-throughput sequencing of amplicon and metagenome was performed to obtain the microbiome profiles and functional traits in P. notoginseng rhizosphere across a large spatial scale. We obtained 639 bacterial and 310 fungal core OTUs, which were mainly affected by soil pH and organic matter (OM). The core taxa were grouped into four ecological clusters (i.e. high pH, low pH, high OM and low OM) for sharing similar habitat preferences. Furthermore, structural equation modelling (SEM) and correlation analyses revealed that the diversity and composition of core microbiomes, as well as the metagenome-derived microbial functions, were related to belowground biomass and saponin contents. Key microbial genera related to the two plant indicators were also identified. In short, this study explored the main driving environmental factors of core microbiomes in the P. notoginseng rhizosphere and revealed that the core microbiomes and microbial functions potentially contributed to the belowground biomass and saponin contents of the plant. This work may enhance our understanding of interactions between microbes and perennial plants and improve our ability to manage root microbiota for the sustainable production of herbal medicine.

Root-Associated Microbiomes of Panax notoginseng under the Combined Effect of Plant Development and Alpinia officinarum Hance Essential Oil.

Essential oils (EOs) have been proposed as an alternative to conventional pesticides to inhibit fungal pathogens. However, the application of EOs is considerably limited due to their highly volatile nature and unpredictable effects on other microbes. In our study, the composition of bacterial and fungal communities from the rhizosphere soil of P. notoginseng under four treatment levels of Alpinia officinarum Hance EO was characterized over several growth stages. Leaf weight varied dramatically among the four EO treatment levels after four months of growth, and the disease index at a low concentration (0.14 mg/g) of EO addition was the lowest among the P. notoginseng growth stages. The content of monomeric saponins was elevated when EO was added. Bacterial and fungal diversity in the absence of plants showed a decreasing trend with increasing levels of EO. Bacterial diversity recovery was more correlated with plant growth than was fungal diversity recovery. Compared with the control (no EO addition), a low concentration of EO significantly accumulated Actinomycota, including Acidothermus, Blastococcus, Catenulispora, Conexibacter, Rhodococcus, and Sinomonas, after one month of plant-microbial interaction. Overall, the results showed that both the plant growth stage and EOs drive changes in the microbial community composition in the rhizosphere of P. notoginseng. Plant development status had a stronger influence on bacterial diversity than on fungal diversity. EO had a more significant effect on fungal community composition, increasing the dominance of Ascomycota when EO concentration was increased. Under the interaction of P. notoginseng growth and EO, a large number of bacterial genera that have been described as plant growth-promoting rhizobacteria (PGPR) responded positively to low concentrations of EO application, suggesting that EO may recruit beneficial microbes in the root zone to cope with pathogens and reduce root rot disease. These results offer novel insights into the relationship between EO application, altered microbial communities in the plant roots, plant growth stage, and disease occurrence.

Biochar increases Panax notoginseng's survival under continuous cropping by improving soil properties and microbial diversity.

Replant problem is widespread in agricultural production and causes serious economic losses, which has limited sustainable cultivation of Panax notoginseng (PN), a well-known medicinal plant in Asia. Here we conducted a field experiment to investigate the effectiveness and possible mechanisms of biochar to improve its survival under continuous cropping. Biochar from tobacco stems was applied at 4 rates of 9.0, 12, 15, and 18 t/ha to a soil where PN has been continuously cultivated for 10 years. After 18 months, soil properties, 5 allelochemicals, including p-hydroxybenzoic acid, vanillic acid, syringic acid, p-coumaric acid, and ferulic acid, key pathogen Fusarium oxysporum, microbial community, and PN survival rate were investigated. Our results show that 10 years' continuous PN cropping led to soil acidification, accumulation of NH4+-N and F. oxysporum, and low PN survival rate. However, biochar increased its survival rate from 6.0% in the control to 69.5% under 15 t/ha treatment. Moreover, soil pH, available P and K, organic matter content, and microbial diversity were increased while NH4+-N and allelochemicals vanillic acid and syringic acid contents were decreased under biochar treatment (P<0.05). Soil available K increased from 177 to 283 mg·kg-1 while NH4+-N decreased from 6.73 to 4.79 mg·kg-1 under 15 t/ha treatment. Further, soil pH, available P and K, and microbial diversity (bacteria and fungi) were positively correlated with PN survival rate, however, NH4+-N content was negatively correlated (P<0.05). Our study indicates that biochar effectively increased the survival rate of Panax notoginseng under continuous cropping by improving soil properties and microbial diversity.

Moisture Controls the Suppression of Panax notoginseng Root Rot Disease by Indigenous Bacterial Communities.

Harnessing indigenous soil microbial suppression is an emerging strategy for managing soilborne plant diseases. Soil moisture is a vital factor in soil microbiomes, but its role in the regulation of microbial suppression is poorly understood. Here, we investigated the correlation of root rot disease of Panax notoginseng with rhizosphere microbial communities mediated by soil moisture gradients from 55% to 100% field capacity (FC); then, we captured the disease-suppressive and disease-inductive microbiomes and validated their functions by a culture experiment with synthetic microbiotas containing keystone species. We found that proper soil moisture at 75% to 95% FC could maintain a disease-suppressive microbiome to alleviate root rot disease. However, extremely low or high soil moistures (>95% FC or <75% FC) could aggravate root rot disease by depleting the disease-suppressive microbiome while enriching the disease-inductive microbiome. Both the low-soil-moisture-enriched pathogen Monographella cucumerina and the high-soil-moisture-enriched pathogen Ilyonectria destructans could synergize with different disease-inductive microbiomes to aggravate disease. Metagenomic data confirmed that low- and high-moisture conditions suppressed antibiotic biosynthesis genes but enriched pathogenicity-related genes, resulting in a change in the soil state from disease suppressive to inductive. This study highlights the importance of soil moisture when indigenous microbial suppression is harnessed for disease control. IMPORTANCE Soilborne diseases pose a major problem in high-intensity agricultural systems due to the imbalance of microbial communities in soil, resulting in the buildup of soilborne pathogens. Harnessing indigenous soil microbial suppression is an emerging strategy for overcoming soilborne plant diseases. In this study, we showed that soil moisture is a key factor in balancing microbiome effects on root rot disease. Proper soil moisture management represent an effective approach to maintain microbial disease resistance by enriching disease-suppressive microbiomes. Conversely, moisture stresses may enrich for a disease-inductive microbiome and aid accumulation of host-specific soilborne pathogens threatening crop production. This work could provide a new strategy for sustainable control of soilborne diseases by enriching the indigenous disease-suppressive microbiome through soil moisture management.

Characteristics and diversity of endophytic bacteria in Panax notoginseng under high temperature analysed using full-length 16S rRNA sequencing.

Panax notoginseng is a traditional Chinese medicinal herb with diverse properties that is cultivated in a narrow ecological range because of its sensitivity to high temperatures. Endophytic bacteria play a prominent role in plant response to climate warming. However, the endophytic bacterial structures in P. notoginseng at high temperatures are yet unclear. In the present study, the diversity and composition of the endophytic bacterial community, and their relationships with two P. notoginseng plants with different heat tolerance capacities were compared using the full-length 16S rRNA PacBio sequencing system. The results revealed that the diversity and richness of endophytic bacteria were negatively associated with the heat tolerance of P. notoginseng. Beneficial Cyanobacteria, Rhodanobacter and Sphingomonas may be recruited positively by heat-tolerant plants, while higher amounts of adverse Proteobacteria such as Cellvibrio fibrivorans derived from soil destructed the cellular protective barriers of heat-sensitive plants and caused influx of pathogenic bacteria Stenotrophomonas maltophilia. Harmonious and conflicting bacterial community was observed in heat-tolerant and heat-sensitive P. notoginseng, respectively, based on the co-occurrence network. Using functional gene prediction of metabolism, endophytic bacteria have been proposed to be symbiotic with host plants; the bacteria improved primary metabolic pathways and secondary metabolite production of plants, incorporated beneficial endophytes, and combated adverse endophytes to prompt the adaptation of P. notoginseng to a warming environment. These findings provided a new perspective on the function of endophytes in P. notoginseng adaptation to high temperatures, and could pave the way for expanding the cultivable range of P. notoginseng.

Appropriate Soil Heat Treatment Promotes Growth and Disease Suppression of Panax notoginseng by Interfering with the Bacterial Community.

In our greenhouse experiment, soil heat treatment groups (50, 80, and 121°C) significantly promoted growth and disease suppression of Panax notoginseng in consecutively cultivated soil (CCS) samples (p < 0.01), and 80°C worked better than 50°C and 121°C (p < 0.01). Furthermore, we found that heat treatment at 80°C changes the microbial diversity in CCS, and the inhibition ratios of culturable microorganisms, such as fungi and actinomycetes, were nearly 100%. However, the heat-tolerant bacterial community was preserved. The 16S rRNA gene and internal transcribed spacer (ITS) sequencing analyses indicated that the soil heat treatment had a greater effect on the Chao1 index and Shannon's diversity index of bacteria than fungi, and the relative abundances of Firmicutes and Proteobacteria were significantly higher than without heating (80 and 121°C, p < 0.05). Soil probiotic bacteria, such as Bacillus (67%), Sporosarcina (9%), Paenibacillus (6%), Paenisporosarcina (6%), and Cohnella (4%), remained in the soil after the 80°C and 121°C heat treatments. Although steam increased the relative abundances of most of the heat-tolerant microbes before sowing, richness and diversity gradually recovered to the level of CCS, regardless of fungi or bacteria, after replanting. Thus, we added heat-tolerant microbes (such as Bacillus) after steaming, which reduced the relative abundance of pathogens, recruited antagonistic bacteria, and provided a long-term protective effect compared to the steaming and Bacillus alone (p < 0.05). Taken together, the current study provides novel insight into sustainable agriculture in a consecutively cultivated system.

Control of pathogenic fungi on Panax notoginseng by volatile oils from the food ingredients Allium sativum and Foeniculum vulgare.

To screen natural drugs with strong inhibitory effects against pathogenic fungi related to P. notoginseng, the antifungal activities of garlic and fennel EOs were studied by targeting P. notoginseng disease-associated fungi, and the possible action mechanisms of garlic and fennel EOs as plant fungicides were preliminarily discussed. At present, the antifungal mechanism of EOs has not been fully established. Therefore, understanding the antifungal mechanism of plant EOs is helpful to address P. notoginseng diseases continuous cropping disease-related obstacles and other agricultural cultivation problems. First, the Oxford cup method and chessboard were used to confirm that the EOs and oxamyl had a significant inhibitory effect on the growth of Fusarium oxysporum. F. oxysporum is the main pathogen causing root rot of P. notoginseng and the preliminary study on the antifungal mechanisms of the EOs against F. oxysporum showed that the inhibition of EOs mainly affects cell membrane permeability and cell processes and affects the enzyme activities of micro-organism, to achieve antifungal effects. Finally, an in vivo model verified that both two EOs could significantly inhibit the occurrence of root rot caused by F. oxysporum.

Purification and characterization of anti-phytopathogenic fungi angucyclinone from soil-derived Streptomyces cellulosae.

Actinomycete strain YIM PH20352, isolated from the rhizosphere soil sample of Panax notoginseng collected in WenShang, Yunnan Province, China, exhibited antifungal activity against some phytopathogenic fungi. The structures of bioactive molecules, isolated from the ethyl acetate extract of the fermentation broth of the strain, were identified as rabelomycin (1) and dehydrorabelomycin (2) based on extensive spectroscopic analyses. Compound 1 exhibited antifungal activity against four tested root-rot pathogens of the Panax notoginseng including Plectosphaerella cucumerina, Alternaria panax, Fusarium oxysporum, and Fusarium solani with the MIC values at 32, 64, 128, and 128 µg/mL, respectively. Compound 2 exhibited antifungal activity against F. oxysporum, P. cucumerina, F. solani, and A. panax with the MIC values at 64, 64, 128, and 128 µg/mL, respectively. Based on the phylogenetic analyses, the closest phylogenetic relative of strain YIM PH20352 is Streptomyces cellulosae NBRC 13027 T (AB184265) (99.88%), so strain YIM PH20352 was identified as Streptomyces cellulosae. To the best of our knowledge, this is the first report of rabelomycin and rabelomycin-type antibiotics from Streptomyces cellulosae and their antifungal activity against root-rot pathogens of the Panax notoginseng.

Autotoxin Rg1 Induces Degradation of Root Cell Walls and Aggravates Root Rot by Modifying the Rhizospheric Microbiome.

Management of crop root rot disease is one of the key factors in ensuring sustainable development in agricultural production. The accumulation of autotoxins and pathogens in soil has been reported as a primary driver of root rot diseases; however, less is known about the correlation of plants, their associated pathogens and microbiome mediated by autotoxins as well as the contributions autotoxins make to the occurrence of root rot disease. Here, we integrated metabolomic, transcriptomic, and rhizosphere microbiome analyses to identify the root cell wall degradants cellobiose and d-galacturonic acid as being induced by the autotoxic ginsenoside Rg1 of Panax notoginseng, and we found that exogenous cellobiose and d-galacturonic acid in addition to Rg1 could aggravate root rot disease by modifying the rhizosphere microbiome. Microorganisms that correlated positively with root rot disease were enriched and those that correlated negatively were suppressed by exogenous cellobiose, d-galacturonic acid, and Rg1. In particular, they promoted the growth and infection of the soilborne pathogen Ilyonectria destructans by upregulating pathogenicity-related genes. Cellobiose showed the highest ability to modify the microbiome and enhance pathogenicity, followed by Rg1 and then d-galacturonic acid. Collectively, autotoxins damaged root systems to release a series of cell wall degradants, some of which modified the rhizosphere microbiome so that the host plant became more susceptible to root rot disease. IMPORTANCE The accumulation of autotoxins and pathogens in soil has been reported as a primary driver of root rot disease and one of the key factors limiting sustainable development in agricultural production. However, less is known about the correlation of plants, their associated pathogens, and the microbiome mediated by autotoxins, as well as the contributions autotoxins make to the occurrence of root rot disease. In our study, we found that autotoxins can damage root systems, thus releasing a series of cell wall degradants, and both autotoxins and the cell wall degradants they induce could aggravate root rot disease by reassembling the rhizosphere microbiome, resulting in the enrichment of pathogens and microorganisms positively related to the disease but the suppression of beneficial microorganisms. Deciphering this mechanism among plants, their associated pathogens, and the microbiome mediated by autotoxins will advance our fundamental knowledge of and ability to degrade autotoxins or employ microbiome to alleviate root rot disease in agricultural systems.

Chemical Constituents of the Essential oil from Cuminum cyminum L. and Its Antifungal Activity against Panax notoginseng Pathogens.

Cuminum cyminum L. (Cumin) is a flavoring agent that is commonly used worldwide, and is rich in essential oil. Essential oils (Eos) have been intensively investigated in regard to their potential for disease control in plants, which is provided a chance for the blossom of green pesticides. The chemical components of Cumin essential oil (CEO) were revealed by GC/MS, such as cuminaldehyde (44.53 %), p-cymene (12.14 %), (-)-ß-pinene (10.47 %) and γ-terpinene (8.40 %), and found they can inhibit the growth of P. notoginseng-associated pathogenic fungi in vitro and the inhibitory effect of cuminaldehyde was similar to that of hymexazol. SEM and TEM images demonstrated that cuminaldehyde and CEO increased cell permeability and disrupted membrane integrity. The expression of disease-related genes of Fusarium oxysporum showed that CEO induced the expression of most genes, which disrupted biosynthesis, metabolism and signaling pathways. These studies verified the potential of CEO as a plant fungicide that is environmentally friendly and provided ideas for developing new products for controlling root diseases that affect P. notoginseng.

Enrichment of Burkholderia in the Rhizosphere by Autotoxic Ginsenosides to Alleviate Negative Plant-Soil Feedback.

The accumulation of autotoxins and soilborne pathogens in soil was shown to be the primary driver of negative plant-soil feedback (NPSF). There is a concerted understanding that plants could enhance their adaptability to biotic or abiotic stress by modifying the rhizosphere microbiome. However, it is not clear whether autotoxins could enrich microbes to degrade themselves or antagonize soilborne pathogens. Here, we found that the microbiome degraded autotoxic ginsenosides, belonging to triterpenoid glycosides, and antagonized pathogens in the rhizosphere soil of Panax notoginseng (sanqi). Deep analysis by 16S rRNA sequencing showed that the bacterial community was obviously changed in the rhizosphere soil and identified the Burkholderia-Caballeronia-Paraburkholderia (BCP) group as the main ginsenoside-enriched bacteria in the rhizosphere soil. Eight strains belonging to the BCP group were isolated, and Burkholderia isolate B36 showed a high ability to simultaneously degrade autotoxic ginsenosides (Rb1, Rg1, and Rd) and antagonize the soilborne pathogen Ilyonectria destructans. Interestingly, ginsenosides could stimulate the growth and biofilm formation of B36, eventually enhancing the antagonistic ability of B36 to I. destructans and the colonization ability in the rhizosphere soil. In summary, autotoxic ginsenosides secreted by P. notoginseng could enrich beneficial microbes in the rhizosphere to simultaneously degrade autotoxins and antagonize pathogen, providing a novel ecological strategy to alleviate NPSF. IMPORTANCE Autotoxic ginsenosides, secreted by sanqi into soil, could enrich Burkholderia sp. to alleviate negative plant-soil feedback (NPSF) by degrading autotoxins and antagonizing the root rot pathogen. In detail, ginsenosides could stimulate the growth and biofilm formation of Burkholderia sp. B36, eventually enhancing the antagonistic ability of Burkholderia sp. B36 to a soilborne pathogen and the colonization of B36 in soil. This ecological strategy could alleviate NPSF by manipulating the rhizosphere microbiome to simultaneously degrade autotoxins and antagonize pathogen.

Myrothins A-F from Endophytic Fungus Myrothecium sp. BS-31 Harbored in Panax notoginseng.

Endophytic fungi play important roles for host's stress tolerance including invasion by pathogenic microbes. Small molecules are common weapons in the microbe-microbe interactions. Panax notoginseng is a widely used traditional Chinese medicinal plant and harbors many endophytes, some exert functions against pathogens. Here, we report six new compounds named myrothins A-F (1-6) produced by Myrothecium sp. BS-31, an endophyte isolated from P. notoginseng, and their antifungal activities against pathogenic fungi causing host root-rot disease. Their structures were elucidated with analysis of spectroscopic data including 1D and 2D NMR, HR-ESI-MS. Myrothins B (2) and E (5) showed the weak activity against Fusarium oxysporum and Phoma herbarum, and myrothins F (6) showed weak activity against F. oxysporum.

Inhibitory effects of essential oils from Asteraceae plant against pathogenic fungi of Panax notoginseng.

AIMS: Diseases caused by pathogenic fungi was a major constrain in increasing productivity and improving quality of Panax notoginseng. The aim of this research was to evaluate the inhibitory activity of essential oils (EOs) from Asteraceae family, Chrysanthemum indicum and Laggera pterodonta, against pathogenic fungi of P. notoginseng. METHODS AND RESULTS: The antifungal activity was investigated using multiple methods, disclosing that the EOs from C. indicum and L. pterodonta are active against hypha growth of different fungi but with different degrees of potency. Checkerboard testing indicated that the combination of EOs with hymexazol had synergistic effect against Pythium aphanidermatum, and exhibited additive effects against bulk of targeted pathogenic fungi. Besides, we found that the baseline sensitivity of Fusarium oxysporum to L. pterodonta EOs was higher than those of C. indicum by means of mycelium growth rate method. Finally, the practicability of those EOs as plant pesticide was confirmed by in vivo model showing that EOs can significantly inhibit the occurrence of root rot of P. notoginseng caused by F. oxysporum. CONCLUSION: Those studies suggest that the EOs from C. indicum and L. pterodonta had the potential to develop into new pollution-free pesticides for the protection of precious Chinese herbal medicines. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provided a new way of biological control for overcoming the frequent diseases occurrence of P. notoginseng.

Insights into Streptomyces spp. isolated from the rhizospheric soil of Panax notoginseng: isolation, antimicrobial activity and biosynthetic potential for polyketides and non-ribosomal peptides.

BACKGROUND: Streptomycetes from the rhizospheric soils are a rich resource of novel secondary metabolites with various biological activities. However, there is still little information related to the isolation, antimicrobial activity and biosynthetic potential for polyketide and non-ribosomal peptide discovery associated with the rhizospheric streptomycetes of Panax notoginseng. Thus, the aims of the present study are to (i) identify culturable streptomycetes from the rhizospheric soil of P. notoginseng by 16S rRNA gene, (ii) evaluate the antimicrobial activities of isolates and analyze the biosynthetic gene encoding polyketide synthases (PKSs) and nonribosomal peptide synthetases (NRPSs) of isolates, (iii) detect the bioactive secondary metabolites from selected streptomycetes, (iv) study the influence of the selected isolate on the growth of P. notoginseng in the continuous cropping field. This study would provide a preliminary basis for the further discovery of the secondary metabolites from streptomycetes isolated from the rhizospheric soil of P. notoginseng and their further utilization for biocontrol of plants. RESULTS: A total of 42 strains representing 42 species of the genus Streptomyces were isolated from 12 rhizospheric soil samples in the cultivation field of P. notoginseng and were analyzed by 16S rRNA gene sequencing. Overall, 40 crude cell extracts out of 42 under two culture conditions showed antibacterial and antifungal activities. Also, the presence of biosynthesis genes encoding type I and II polyketide synthase (PKS I and PKS II) and nonribosomal peptide synthetases (NRPSs) in 42 strains were established. Based on characteristic chemical profiles screening by High Performance Liquid Chromatography-Diode Array Detector (HPLC-DAD), the secondary metabolite profiles of strain SYP-A7257 were evaluated by High Performance Liquid Chromatography-High Resolution Mass Spectrometry (HPLC-HRMS). Finally, four compounds actinomycin X2 (F1), fungichromin (F2), thailandin B (F7) and antifungalmycin (F8) were isolated from strain SYP-A7257 by using chromatography techniques, UV, HR-ESI-MS and NMR, and their antimicrobial activities against the test bacteria and fungus were also evaluated. In the farm experiments, Streptomyces sp. SYP-A7257 showed healthy growth promotion and survival rate improvement of P. notoginseng in the continuous cropping field. CONCLUSIONS: We demonstrated the P. notoginseng rhizospheric soil-derived Streptomyces spp. distribution and diversity with respect to their metabolic potential for polyketides and non-ribosomal peptides, as well as the presence of biosynthesis genes PKS I, PKS II and NRPSs. Our results showed that cultivatable Streptomyces isolates from the rhizospheric soils of P. notoginseng have the ability to produce bioactive secondary metabolites. The farm experiments suggested that the rhizospheric soil Streptomyces sp. SYP-A7257 may be a potential biological control agent for healthy growth promotion and survival rate improvement of P. notoginseng in the continuous cropping field.

Trichoderma panacis sp. nov., an endophyte isolated from Panax notoginseng.

An endophytic member of the genus Trichoderma was isolated from the root of a healthy 3-year-old Panax notoginseng in Yunnan province, PR China. The results of phylogenetic analyses based on a combined of ITS, tef1 and rpb2 indicated that this isolate was distinct from other species of the genus Trichoderma and closely related to Trichoderma songyi. It can be distinguished from T. songyi by its slower growth rates on PDA and colony morphology. The novel isolate formed conidia in thick white pustules scattered mostly at the margin. Its conidiophores tended to be regularly verticillium-like, little branched, sometimes substituted by phialides singly or in whorls. Conidia are smooth, mostly broadly subglobose to ellipsoidal. In combination with the genotypic and phenotypic characteristics, all data demonstrated that the fungus studied represented a unique and distinguishable novel species of the genus Trichoderma, for which the name Trichoderma panacis sp. nov. is proposed.

Microbial Community Changes in the Rhizosphere Soil of Healthy and Rusty Panax ginseng and Discovery of Pivotal Fungal Genera Associated with Rusty Roots.

Panax ginseng Meyer, a valuable medicinal plant, is severely threatened by rusty root, a condition that greatly affects its yield and quality. Studies investigating the relationship between soil microbial community composition and rusty roots are vital for the production of high-quality ginseng. Here, high-throughput sequencing was employed to systematically characterize changes in the soil microbial community associated with rusty roots. Fungal diversity was lower in the soils of rusty root-affected P. ginseng than in those of healthy plants. Importantly, principal coordinate analysis separated the fungal communities in the rhizosphere soils of rusty root-affected ginseng from those of healthy plants. The dominant bacterial and fungal genera differed significantly between rhizosphere soils of healthy and rusty root-affected P. ginseng, and linear discriminant analysis effect size (LEfSe) further indicated a strong imbalance in the soil microbial community of diseased plants. Significantly enriched bacterial genera (including Rhodomicrobium, Knoellia, Nakamurella, Asticcacaulis, and Actinomadura) were mainly detected in the soil of rusty root-affected P. ginseng, whereas significantly enriched fungal genera (including Xenopolyscytalum, Arthrobotrys, Chalara, Cryptococcus, and Scutellinia) were primarily detected in the soil of healthy plants. Importantly, five fungal genera (Cylindrocarpon, Acrophialophora, Alternaria, Doratomyces, and Fusarium) were significantly enriched in the soil of rusty root-affected plants compared with that of healthy plants, suggesting that an increase in the relative abundance of these pathogenic fungi (Cylindrocarpon, Alternaria, and Fusarium) may be associated with ginseng rusty roots. Additionally, this study is the first to report that an increase in the relative abundances of Acrophialophora and Doratomyces in the rhizosphere of P. ginseng may be associated with the onset of rusty root symptoms in this plant. Our findings provide potentially useful information for developing biological control strategies against rusty root, as well as scope for future screening of fungal pathogens in rusty roots of P. ginseng.

Autotoxic Ginsenoside Disrupts Soil Fungal Microbiomes by Stimulating Potentially Pathogenic Microbes.

Autotoxic ginsenosides have been implicated as one of the major causes for replant failure of Sanqi ginseng (Panax notoginseng); however, the impact of autotoxic ginsenosides on the fungal microbiome, especially on soilborne fungal pathogens, remains poorly understood. In this study, we aimed to investigate the influence of the ginsenoside monomers Rg1, Rb1, and Rh1, and that of their mixture (Mix), on the composition and diversity of the soil fungal community, as well as on the abundance and growth of the soilborne pathogen Fusarium oxysporum in pure culture. The addition of autotoxic ginsenosides altered the composition of the total fungal microbiome, as well as the taxa within the shared and unique treatment-based components, but did not alter alpha diversity (α-diversity). In particular, autotoxic ginsenosides enriched potentially pathogenic taxa, such as Alternaria, Cylindrocarpon, Gibberella, Phoma, and Fusarium, and decreased the abundances of beneficial taxa such as Acremonium, Mucor, and Ochroconis Relative abundances of pathogenic taxa were significantly and negatively correlated with those of beneficial taxa. Among the pathogenic fungi, the genus Fusarium was most responsive to ginsenoside addition, with the abundance of Fusarium oxysporum consistently enhanced in the ginsenoside-treated soils. Validation tests confirmed that autotoxic ginsenosides promoted mycelial growth and conidial germination of the root rot pathogen F. oxysporum In addition, the autotoxic ginsenoside mixture exhibited synergistic effects on pathogen proliferation. Collectively, these results highlight that autotoxic ginsenosides are capable of disrupting the equilibrium of fungal microbiomes through the stimulation of potential soilborne pathogens, which presents a significant hurdle in remediating replant failure of Sanqi ginseng.IMPORTANCE Sanqi ginseng [Panax notoginseng (Burk.) F. H. Chen] is geoauthentically produced in a restricted area of southwest China, and successful replanting requires a rotation cycle of more than 15 to 30 years. The increasing demand for Sanqi ginseng and diminishing arable land resources drive farmers to employ consecutive monoculture systems. Replant failure has severely threatened the sustainable production of Sanqi ginseng and causes great economic losses annually. Worse still, the acreage and severity of replant failure are increased yearly, which may destroy the Sanqi ginseng industry in the near future. The significance of this work is to decipher the mechanism of how autotoxic ginsenosides promote the accumulation of soilborne pathogens and disrupt the equilibrium of soil fungal microbiomes. This result may help us to develop effective approaches to successfully conquer the replant failure of Sanqi ginseng.

(±)-Preisomide: A new alkaloid featuring a rare naturally occurring tetrahydro-2H-1,2-oxazin skeleton from an endophytic fungus Preussia isomera by using OSMAC strategy.

A new alkaloid, named (±)-preisomide (1), together with five known polyketides (2-6), were isolated from an endophytic fungus Preussia isomera in Panax notoginseng by using one strain-many compounds (OSMAC) strategy. Their structures were identified by extensive spectroscopic experiments and comparison with literature data. Structurally, compound 1 possessed a rare naturally occurring tetrahydro-2H-1,2-oxazin ring. Compound 6 displayed significant antibacterial activity against multidrug-resistant Enterococcus faecium, methicinllin-resistant Staphylococcus aureus and multidrug-resistant Enterococcus faecalis with an MIC value of 25 µg/mL, as well as moderate antifungal activity against Gibberella saubinetii with an MIC value of 50 µg/mL.

Fungicidal Activity of Essential Oils from Cinnamomum cassia against the Pathogenic Fungi of Panax notoginseng Diseases.

The frequent disease of Panax notoginseng caused by the pathogenic fungi in field cultivation has become the major threaten to the sustainable development of it. The present study was conducted to find natural agent with potential inhibition against pathogen. Therefore, the inhibitory effects of Cinnamomum cassia (L.) J.Presl essential oils (EOs) against P. notoginseng associated pathogenic fungi were conducted both in vitro and in vivo experiments. The results of the Oxford cup test revealed that C. cassia dry bark EO (50 mg/mL) had significant inhibitory activity on the growth of all tested fungi, and the growth of various pathogens was completely inhibited, except for that of Fusarium solani. Therefore, the constituents of C. cassia EOs were analyzed by GC/MS, and the research demonstrated that the main constituents of C. cassia dry bark EO were trans-cinnamaldehyde (75.65 %), (E)-2-methoxycinnamaldehyde (6.08 %), cinnamaldehyde (3.47 %) and cinnamyl acetate (1.02 %). The MIC results showed that C. cassia dry bark EO and the main compounds had good antifungal effect on the tested strains, and the inhibitory effect was similar to that of hymexazol (chemical pesticide). By analyzing the value of the fraction inhibitory concentration index (FICI), additive effects, irrelevant effects and synergistic effects were observed after the mixture of hymexazol against various pathogens. Moreover, in vivo model showed that C. cassia dry bark EO could reduce the occurrence of anthrax in P. notoginseng. To widen the resources of C. cassia available, the compositions of both C. cassia fresh bark and leaf EOs were also tested and many common compositions existed among them. Taken together, it was concluded that C. cassia EO had the potential use in the field to reduce the pathogenic disease.