Pantoea- Bacillus as a Composite Microbial Community: Inhibition and Potential Mechanism Against Potato Anthracnose Disease

Abstract

The potato (Solanum tuberosum), an important component of global food security, often faces threats from various diseases during its growth process, especially potato anthracnose (Colletotrichum coccodes), which severely affects crop yield and quality. In this study, we successfully isolated and identified two bacteria with potential for biological control, (Pantoea agglomerans) and (Bacillus subtilis). The experimental results indicate that the bacterial suspensions of strains JZ-1-1-1 and JZ-2-2-2 had a significant inhibitory effect on the pathogen ZL-7, with the inhibition rate of JZ-1-1-1 reaching as high as 55.21%. The inhibition rate of JZ-2-2-2 was 53.48%. When these two strains were mixed at a 4:6 ratio, the inhibitory effect on pathogenic bacteria was even more significant, reaching 68.58% inhibition. In addition, the composite microbial community produced biofilms with their yield gradually increasing within 24 h and showing a slight decrease after 72 h. The efficacy test further indicated that the composite bacterial suspension was highly effective in controlling the spread of lesions, with an efficacy rate as high as 81.40%. In the analysis of defense enzyme activity, peroxidase (POD) and superoxide dismutase (SOD) levels peaked on day seven, while the composite bacterial suspension significantly reduced malondialdehyde (MDA) and polyphenol oxidase (PPO) activity. Quantitative real-time PCR confirmed that these two strains effectively colonized the surface of potato tubers. In summary, this study provides an important theoretical basis and practical guidance for the application of biological methods for the prevention and control of potato anthracnose.

Keywords: antibacterial activity; composite microbial community; defense enzymes; potato anthracnose; quantitative real-time PCR.

Figure 1

Morphological characteristics and phylogenetic tree of different bacteria. (A) Morphological characteristics of JZ1-1-1 on LB medium; (B) Micromorphology of JZ1-1-1 cells, scale bar = 20 μm; (C) Morphological characteristics of JZ2-2-2 on LB medium; (D) Micromorphology of JZ2-2-2 cells, scale bar = 20 μm; (E): Phylogenetic tree of JZ1-1-1 constructed based on the sequences of 16S rDNA, gyrB, frr, and rpoD genes. This tree was constructed using the neighbor-joining method, and there 1000 lead repetitions show a lead rate greater than 50%. (F) Phylogenetic tree of JZ2-2-2 constructed based on the sequences of 16S rDNA, atpD, gyrA, and rpoB genes. This tree was constructed using the neighbor-joining method, and there 1000 lead repetitions show a lead rate greater than 50%.

Figure 2

Antimicrobial activity of different fermentation products against Colletotrichum coccodes. (A) Antimicrobial activity of JZ1-1-1 bacterial suspension against Colletotrichum coccodes; (B) Antimicrobial activity of JZ2-2-2 bacterial suspension against Colletotrichum coccodes; (C) Colletotrichum coccodes ZL-7; (D) Antimicrobial activity of JZ1-1-1 fermentation broth against Colletotrichum coccodes; (E) Antimicrobial activity of JZ2-2-2 fermentation broth against Colletotrichum coccodes.

Figure 3

Antifungal activity of different ratios of JZ1-1-1 and JZ2-2-2 composite bacterial suspensions against Colletotrichum coccodes.

Figure 4

Biofilm formation ability of the composite microbial community at 28 °C. (A) Biofilm morphology of the composite microbial community at 0–96 h (B) 0 h (C) 48 h (D) 96 h. (E) 0, 48, and 96-h states of crystal violet-stained biofilms at different time intervals.

Figure 5

The condition of potato tubers at different growth stages. Note: (T): Potato Pathogen ZL-7, (S): Control, (X): Composite Microbial Community, (M): Prochloraz Mode, (Y): Preventive Mode, (Z): Therapeutic Mode.

Figure 6

The effect of the composite microbial community on potato-related defense enzymes. Note: (A) POD; (B) SOD; (C) MDA; (D) PPO. Note: (T): Potato Pathogen ZL-7, (S): Control, (X): Composite Microbial Community, (M): Pro-chloraz Mode, (Y): Preventive Mode, (Z): Therapeutic Mode. Different lowercase letters represent the differences between different treatments (p < 0.05).

Figure 7

Electrophoresis results of related primers amplification for strains JZ1-1-1 and JZ2-2-2. Note: M: DNA Marker 500; 1: JZ1-1-1; 2: JZ2-2-2; 3: Bacillus amyloliq-uefaciens QS10-6; 4: Bacillus atrophaeus QS2-5; 5: Bacillus velezensis QS2-13; 6: Bacillus safensis BM-7; 7: Bacillus atrophaeus Q2-7; 8: JZ2-2-2; 9: JZ1-1-1; 10: Bacillus amyloliq-uefaciens QS10-6; 11: Bacillus atrophaeus QS2-5; 12: Bacillus velezensis QS2-13; 13: Bacillus safensis BM-7; 14: Bacillus atrophaeus QB-2-7.

Figure 8

Fluorescent quantification and colonization detection of different strains. (A) PCR Dissociation Curve of Strain JZ1-1-1.The curves of different colors represent the dissolution curve of strain JZ1-1-1. (B) Amplification Curve of Strain JZ1-1-1, the curves of different colors represent the amplification curve of strain JZ1-1-1. (C) PCR Dissociation Curve of Strain JZ2-2-2 The curves of different colors represent the dissolution curve of strain JZ2-2-2. (D) Amplification Curve of Strain JZ2-2-2, the curves of different colors represent the amplification curve of strain JZ2-2-2 (E) Curve Equation Established by Ct Value (Y) and DNA Concentration (X) of Strain JZ1-1-1, the dotted line represents the trend line of strain JZ1-1-1. (F) Curve Equation Established by Ct Value (Y) and DNA Concentration (X) of Strain JZ2-2-2, the dotted line represents the trend line of strain JZ2-2-2. (G) Detection of colonization levels of strain JZ 1-1-1 and strain JZ 2-2-2 in potato tubers. Different lowercase letters represent the differences between different treatments (p < 0.05).