A bacterial strain JI39 that had plant growth-promoting traits was isolated from the rhizosphere soil of Panax ginseng. It had the ability to produce high indole-3-acetic acid (13.1 μg/ml), phosphate solubilization (164.2 μg/ml), potassium solubilization (16.1 μg/ml), and nitrogen fixation. The strain JI39 was identified to be Arthrobacter nicotinovorans based on morphological, physiological, and biochemical traits and through 16S rDNA sequence analysis. The optimal culture environment for strain growth was 1.0% NaCl, 30°C, pH 6.0, and without UV irradiation. The strain can produce cellulase and protease. The strain JI39 can significantly promote the growth of ginseng. After ginseng seeds were treated with 3 × 10⁸ CFU/ml of JI39 bacterial suspension, the shoot's length was significantly increased by 64.61% after 15 days. Meanwhile, the fresh weight of 2-year-old ginseng roots was significantly increased by 24.70% with a treatment by the 10⁸ CFU/ml bacterial suspension after 150 days in the field. The gene expression of phenylalanine ammonia-lyase (PAL), β-1.3 glucanase (β-1,3-GA), chitinase (CHI), superoxide dismutase (SOD), and peroxidase (POD) of ginseng was upregulated, and it also can improve the soil urease, phosphatase, invertase, and catalase activity. In conclusion, the bacterial strain JI39 could efficiently promote the growth of ginseng and has the potential to be a good microbial fertilizer for ginseng.

(1) Background: Ilyonectria robusta can cause ginseng to suffer from rusty root rot. Secondary metabolites (SMs) produced by Bacillus methylotrophicus NJ13 can inhibit the mycelial growth of I. robusta. However, the molecular mechanism of the inhibition and response remains unclear. (2) Methods: Through an in vitro trial, the effect of B. methylotrophicus NJ13’s SMs on the hyphae and conidia of I. robusta was determined. The change in the physiological function of I. robusta was evaluated in response to NJ13’s SMs by measuring the electrical conductivity, malondialdehyde (MDA) content, and glucose content. The molecular interaction mechanism of I. robusta’s response to NJ13’s SMs was analyzed by using transcriptome sequencing. (3) Results: NJ13’s SMs exhibited antifungal activity against I. robusta: namely, the hyphae swelled and branched abnormally, and their inclusions leaked out due to changes in the cell membrane permeability and the peroxidation level; the EC₅₀ value was 1.21% (v/v). In transcripts at 4 dpi and 7 dpi, the number of differentially expressed genes (DEGs) (|log₂(fold change)| > 1, p adj ≤ 0.05) was 1960 and 354, respectively. NJ13’s SMs affected the glucose metabolism pathway, and the sugar-transporter-related genes were downregulated, which are utilized by I. robusta for energy production. The cell wall structure of I. robusta was disrupted, and chitin-synthase-related genes were downregulated. (4) Conclusions: A new dataset of functional responses of the ginseng pathogenic fungus I. robusta was obtained. The results will benefit the development of targeted biological fungicides for I. robusta and the study of the molecular mechanisms of interaction between biocontrol bacteria and phytopathogenic fungi.

The Bacillus velezensis strain NT35, which has strong biocontrol ability, was isolated from the rhizosphere soil of Panax ginseng. The antifungal effects of the NT35 strain against the mycelium and spore growth of Ilyonectria robusta, which causes ginseng rusty root rot, were determined. The inhibitory rate of I. robusta mycelial growth was 94.12% when the concentration of the NT35 strain was 10⁷ CFU·mL⁻¹, and the inhibitory rates of I. robusta sporulation and spore germination reached 100 and 90.31%, respectively, when the concentration of the NT35 strain was 10⁴ and 10⁸ CFU·mL⁻¹, respectively. Strain NT35 had good prevention effects against ginseng rust rot indoors and in the field with the control effect 51.99%, which was similar to that of commercial chemical and biocontrol agents. The labeled strain NT35-Rif¹⁶⁰-Stre⁴⁰⁰ was obtained and colonized ginseng roots, leaves, stems and rhizosphere soil after 90 days. Bacillus velezensis NT35 can induce a significant increase in the expression of five defensive enzyme-encoding genes and ginsenoside biosynthesis-related genes in ginseng. In the rhizosphere soil, the four soil enzymes and the microbial community improved during different periods of ginseng growth in response to the biocontrol strain NT35. The NT35 strain can recruit several beneficial bacteria, such as Luteimonas, Nocardioides, Sphingomonas, and Gemmatimonas, from the rhizosphere soil and reduce the relative abundance of Ilyonectria, Fusarium, Neonectria and Dactylonectria, which cause root rot and rusty root rot in ginseng plants. The disease indices were significantly negatively correlated with the abundances of Sphingomonas and Trichoderma. Additionally, Sphingomonadales, Sphingomonadaceae and actinomycetes were significantly enriched under the NT35 treatment according to LEfSe analysis. These results lay the foundation for the development of a biological agent based on strain NT35.

Rusty root rot is the most destructive soilborne disease of ginseng caused by pathogenic Ilyonectria spp., predominantly Ilyonectria robusta, in China. However, there remains no effective strategy to control the disease. Current control of the disease requires that soil and ginseng seeds and seedlings infected with I. robusta are avoided during planting. Therefore, rapid and accurate detection of I. robusta would be indispensable in disease control programs. A one-step polymerase chain reaction (PCR) and quantitative real-time PCR (qPCR) assay was developed to detect I. robusta in ginseng seeds, roots, and soil. The species-specific primers HIS H3-F and HIS H3-R, designed based on a partial histone gene sequence of I. robusta, yielded a 268-bp product using the optimized PCR and qPCR protocol. DNA of I. robusta was detected by qPCR in all diseased soil and ginseng roots and seeds resulting from artificial inoculation and sampled from natural fields. I. robusta was detected at an abundance of 1.42 fg/μl at 12 h postinoculation and 191.31 fg/μl at 7 days postinoculation in ginseng roots that showed disease symptoms. In naturally infected soil sampled from ginseng fields, pathogen abundances ranging from 13.23 to 503.39 fg/μl were detected, which were 2.04 to 11.01 times higher than those in ginseng roots. The pathogen was first detected and was more abundant on the surface of the ginseng seed coat compared with that in the seed kernel. This study provides a high-efficiency detection technique for early diagnosis of I. robusta and real-time disease prevention and control strategies.

A biocontrol Bacillus velezensis strain, NT35, was isolated from the rhizosphere soil of ginseng, and its sterile filtrate was obtained through a 0.22 μm filter which had a significant inhibitory effect against Ilyonectria robusta, which causes rusty root rot in Panax ginseng. In order to obtain the best sterile filtrate, the medium fermentation conditions of the strain NT35 were optimized using response surface methodology (RSM), and the best composition was obtained. Therefore, the fermentation medium was composed of yeast extract powder 2.5%, cornmeal 1.5%, K₂HPO₄ 1.5%, and (NH₄)₂SO₄ 2.5%. The optimal inoculum amount was 6%, at an initial pH value of 7.0 and culturing at 34 °C at 180 rpm. The antifungal protein 1-4-2F was obtained through precipitation via 30% (NH₄)₂SO₄ precipitation, desalting by Sephadex G-25, ion-exchange chromatography, and gel filtration chromatography. Tricine-SDS-PAGE showed that the purified protein had a relative molecular weight of approximately 6.5 kDa. The protein 1-4-2F was relatively stable and had better antifungal activity at pH 4–10 and 20–100 °C under ultraviolet irradiation of 30 W. The amino acid sequence of protein 1-4-2F was obtained using mass spectrometry, and had 100% similarity to a hypothetical protein from B. velezensis YAU B9601-Y2 (Accession No: AFJ62117). Its molecular weight was 10.176 kDa, the isoelectric point was 9.08, and its sequence coverage reached 49%. The EC₅₀ value of the protein 1-4-2F against I. robusta was 1.519 μg·mL⁻¹. The mycelia morphology of I. robusta changed significantly after treatment with antifungal protein under microscopic observation; the branches of the mycelia increased, distorted, partially swelled into a spherical or elliptical shape, and even ruptured; and the cells became vacuoles.

Ginseng rusty root rot, caused by Ilyonectria robusta, is the most severe soil-borne disease affecting ginseng cultivation. A synthetic microbial community (SynCom) was developed for the first time using antagonistic and growth-promoting endophytic and rhizosphere bacterial strains to control ginseng rusty root rot, and its synergistic mechanisms had been clarified. The 89 out of 98 biocontrol bacterial groups exhibited synergistic inhibitory effects against I. robusta, while 40 consortia showed both growth-promoting and synergistic effects. Among these, the 24 consortia inhibited mycelial growth of I. robusta by more than 80%, and seven consortia suppressed spore growth by over 60%. The inhibition rates were increased by 19%–131% and 5%−137%, respectively, compared with single strain treatment. The biocontrol consortia achieved superior control efficacy against ginseng rusty root rot in the field, which six consortia exhibited control efficacy exceeding 50%, with the consortium CL95 showing the highest efficacy at 66.67% and significantly promoted ginseng growth. The content of I. robusta in ginseng roots decreased by 46.97% following CL95 treatment compared to the single-strain NJ13. Mechanistic studies indicated that the colonization efficiency of the consortia in ginseng tissues and rhizosphere soil was significantly higher than that of single strains within 63 days. The colonization of CL95 in ginseng leaves, stems, epidermis, root core, lateral root, fibrous root and soil was 33%–215% higher than that of single strain NJ13, respectively. In addition, compared with NJ13, the four soil enzyme activities were increased by 100%–150%, and the expression levels of five defense enzyme genes in three different growth periods of ginseng were increased by 75%–167%, among which CL95 had the most obvious effect. It was also found that biocontrol consortia significantly increased the diversity of microbial communities and the abundance of beneficial fungi and bacteria in ginseng rhizosphere soil, while significantly reducing the abundance of pathogenic fungi. Correlation analysis showed that the consortia were significantly positively correlated with root weight and soil enzyme activity, promoting ginseng growth. This study lays a theoretical foundation for overcoming the limitations of single-strain biocontrol agents and highlight the potential for industrial application of biocontrol consortia in sustainable agriculture.