Medium Optimization and Fermentation Kinetics for Antifungal Compounds Production by an Endophytic Paenibacillus polymyxa DS-R5 Isolated from Salvia miltiorrhiza.

An endophytic bacterium Paenibacillus polymyxa DS-R5 which can effectively inhibit the growth of pathogenic fungi was isolated from Salvia miltiorrhiza in our previous study. By using hydrochloric acid precipitation, methanol extraction, silica gel column isolation, dextran gel chromatography column, and HPLC, 3 compounds with antifungal activity were isolated. To further improve the production of antifungal compounds produced by this strain, fermentation medium was optimized using one-factor-at-a-time, Plackett-Burman design, and Box-Behnken design experiments. Through statistical optimization, the optimal medium composition was determined to be as follows: 14.7 g/l sucrose, 20.0 g/l soluble starch, 7.0 g/l corn steep liquor, 10.0 g/l (NH4)2SO4, and 0.7 g/l KH2PO4. In this optimized medium, the highest titer of antifungal compounds reached 3452 U/ml, which was 123% higher than that in the initial medium. In addition, in order to guide scale-up for production, logistic and Luedeking-Piret equations were proposed to predict the cell growth and antifungal compounds production. The fermentation kinetics and empirical equations of the coefficients (X0, Xm, µm, α, and ß) for the two models were reported, which will aid the design and optimization of industrial processes. The degrees of fit between calculated values of the model and the experimental data were 0.989 and 0.973, respectively. The results show that the cell growth and product synthesis models established in this study may better reflect the dynamic process of antifungal compounds production and provide a theoretical basis for further optimization and on-line monitoring of the fermentation process.

Isolation and identification of a new biocontrol bacteria against Salvia miltiorrhiza root rot and optimization of culture conditions for antifungal substance production using response surface methodology.

BACKGROUND: S. miltiorrhiza root rot is a soil-borne disease mainly caused by Fusarium solani and Fusarium oxysporum, which has spread rapidly in China in recent years. To reduce the amount of pesticides to control this plant fungal disease, biological control using endophytic bacteria is a promising method. Many endophytic bacteria show good biocontrol potential against various plant fungal diseases. The aims of this study were to isolate and identify endophytic bacteria with antifungal activity from Salvia miltiorrhiza plant tissue. In order to increase antifungal substances production, the culture conditions of the isolated DS-R5 strain were optimized through response surface methodology. RESULTS: Thirteen endophytic bacteria with antifungal activity against the target pathogenic fungus were successfully screened. The DS-R5 strain that had the strongest antifungal activity was identified based on morphological, physiological and biochemical characteristics, 16S rRNA and gyrB sequence analysis.The results of response surface methodology experiments showed that the optimal values of the three significant factors were as follows: medium volume, 51.0 ml; initial pH, 6.7; fermentation temperature, 33.1 °C. Under these optimal culture conditions, the titer of antifungal substances produced by the DS-R5 strain was 77.6% higher than that under the initial culture conditions. CONCLUSIONS: The antifungal activity of endophytic bacteria from Salvia miltiorrhiza has been demonstrated for the first time, which may benefit future crop quality and production. In addition, response surface methodology can be well applied the optimization of culture conditions for antifungal substance, which lays the foundation for further research on strain DS-R5.

Colonization Characteristics of Poplar Fungal Disease Biocontrol Bacteria N6-34 and the Inhibitory Effect on Pathogenic Fungi by Real-Time Fluorescence Quantitative PCR Detection.

Botryosphaeria dothidea is one of the most important diseases which can cause poplar canker. In our previous study, the endophytic Bacillus subtilis N6-34 screened from poplar tissue was found to be an antagonistic strain against B. dothidea. In order to ascertain the colonization rule of B. subtilis N6-34 in poplar plants, colonization of B. subtilis N6-34 labeled with a green fluorescent protein (GFP) was investigated in poplar plants and the rhizosphere soil. To confirm the inhibitory effect of the strain N6-34 on pathogenic fungi, real-time fluorescent quantitative PCR experiment with Fusarium oxysporum as the target strain was carried out. Firstly, a plasmid (pHT01-P43GFPmut3a) containing gfp gene was successfully transformed into wild B. subtilis N6-34, which has the similar characteristics with the strain N6-34 in cell growth and antifungal activity. The poplar pot experiments were carried out to examine the colonization rules and colonization quantity in poplar plants and rhizosphere soil. Observation with a confocal laser scanning microscope showed that GFP-labeled B. subtilis N6-34 (N6-34-GFP) could colonize in primary root, lateral root and adventitious root. With the extension of inoculation time, the colonization quantity of N6-34-GFP in the rhizosphere soil and poplar plants showed a trend of first increasing, then stabilizing for a period of time and then decreasing. The real-time fluorescent quantitative PCR result showed a gradual decrease in the number of F. oxysporum with increasing inoculation time. Therefore, N6-34-GFP exhibited colonization in the rhizosphere soil and different parts of poplar plants. In addition, the strain N6-34 could inhibit the growth of pathogenic fungi. The ability of B. subtilis N6-34 to colonize in the rhizosphere soil and poplar plants and to inhibit fungal growth in vitro suggest a potential application of this strain as a biological control agent.

Antibacterial and antitumor activity of Bogorol B-JX isolated from Brevibacillus laterosporus JX-5.

Antimicrobial peptides are promising anti-infective agent candidates because they have a broad antimicrobial spectrum and bioactivity and are unlikely to elicit antibiotic resistance. The bogorols represent a new cationic antibiotic peptide and possess great therapeutic potential because of their bioactivity and precise mode of action. Here, we report that Bogorol B-JX (BBJX), a peptide previously isolated from Brevibacillus laterosporus JX-5 by us, has significant antibacterial and antitumor activities in vitro. BBJX was found to inhibit methicillin-resistant Staphylococcus aureus (MRSA) at 2.5 µg/mL with distinct mechanisms of action from those against Bacillus bombyseptieus and Escherichia coli. It penetrates MRSA membrane with little visible destruction and binds to genomic DNA. BBJX could inhibit the proliferation of human histiocytic lymphoma cell line U-937 and ConA-activated spleen cells at 5 µg/mL, but was not cytotoxic to the Jurkat cells, resting spleen cells or differentiated macrophage-like U-937 immunocytes. Moreover, BBJX caused apoptosis of U-937 cells by opening the mitochondrial permeability transition pore and stimulating the production of reactive oxygen species. Taken together, these studies provided basis for future medical application of the bogorols.

Antifungal activity of Brevibacillus laterosporus JX-5 and characterization of its antifungal components.

The establishment of safe and effective methods for controlling fungal disease is an urgent issue in agriculture and forestry. Microbiological control of plant disease is expected to achieve better results than use of chemically derived fungicides. This study aimed to establish Brevibacillus laterosporus JX-5 as a potential microbiological control agent of poplar canker. The bacterium was isolated from the poplar rhizosphere and demonstrated significant growth inhibition of several pathogenic fungi in vitro. The antifungal components of Br. laterosporus JX-5 were isolated and identified. The fermentation broth of Br. laterosporus JX-5 and its main antifungal component, designated as component B, reduced Botryosphaeria dothidea associated canker of the excised poplar branch by 70 and 90%, respectively. Component B is considerably heat-stable, adaptable to a broad pH range, and UV-resistant. It could inhibit Bo. dothidea by permeating the fungal membrane, fracturing the nuclei, damaging the cell wall, and eventually killing the pathogenic fungus. The antifungal activity exhibited by Br. laterosporus JX-5 and its bioactive metabolic products indicate its feasibility as a potential biocontrol agent for plant diseases.

Isolation and characterization of phosphofungi, and screening of their plant growth-promoting activities.

Rhizospheric microorganisms can increase phosphorus availability in the soil. In this regard, the ability of phosphofungi to dissolve insoluble phosphorus compounds is greater than that of phosphate-solubilizing bacteria. The aim of the current study was to identify efficient phosphofungi that could be developed as commercial microbial agents. Among several phosphate-solubilizing fungal isolates screened, strain CS-1 showed the highest phosphorus-solubilization ability. Based on phylogenetic analysis of the internal transcribed spacer region sequence, it was identified as Aspergillus niger. High-performance liquid chromatography analysis revealed that the mechanism of phosphorus solubilization by CS-1 involved the synthesis and secretion of organic acids, mainly oxalic, tartaric, and citric acids. Furthermore, strain CS-1 exhibited other growth-promoting abilities, including efficient potassium release and degradation of crop straw cellulose. These properties help to returning crop residues to the soil, thereby increasing nutrient availability and sustaining organic matter concentration therein. A pot experiment revealed that CS-1 apparently increased the assessed biometric parameters of wheat seedlings, implying the potential of this strain to be developed as a commercial microbial agent. We used Illumina MiSeq sequencing to investigate the microbial community composition in the rhizosphere of uninoculated wheat plants and wheat plants inoculated with the CS-1 strain to obtain insight into the effect of the CS-1 strain inoculation. The data clearly demonstrated that CS-1 significantly reduced the content of pathogenic fungi, including Gibberella, Fusarium, Monographella, Bipolaris, and Volutella, which cause soil-borne diseases in various crops. Strain CS-1 may hence be developed into a microbial agent for plant growth improvement.

Pyrosequencing reveals fungal communities in the rhizosphere of Xinjiang Jujube.

Fungi are important soil components as both decomposers and plant symbionts and play a major role in ecological and biogeochemical processes. However, little is known about the richness and structure of fungal communities. DNA sequencing technologies allow for the direct estimation of microbial community diversity, avoiding culture-based biases. We therefore used 454 pyrosequencing to investigate the fungal communities in the rhizosphere of Xinjiang jujube. We obtained no less than 40,488 internal transcribed spacer (ITS) rDNA reads, the number of each sample was 6943, 6647, 6584, 6550, 6860, and 6904, and we used bioinformatics and multivariate statistics to analyze the results. The index of diversity showed greater richness in the rhizosphere fungal community of a 3-year-old jujube than in that of an 8-year-old jujube. Most operational taxonomic units belonged to Ascomycota, and taxonomic analyses identified Hypocreales as the dominant fungal order. Our results demonstrated that the fungal orders are present in different proportions in different sampling areas. Redundancy analysis (RDA) revealed a significant correlation between soil properties and the abundance of fungal phyla. Our results indicated lower fungal diversity in the rhizosphere of Xinjiang jujube than that reported in other studies, and we hope our findings provide a reference for future research.