Antagonistic Bacillus species as a biological control of ginseng root rot caused by Fusarium cf. incarnatum

Abstract

Background: This study aimed to develop a biocontrol system for ginseng root rot caused by Fusarium cf. incarnatum.

Methods: In total, 392 bacteria isolated from ginseng roots and various soils were screened for their antifungal activity against the fungal pathogen, and a bacterial isolate (B2-5) was selected as a promising candidate for the biocontrol because of the strong antagonistic activity of the bacterial cell suspension and culture filtrate against pathogen.

Results: The bacterial isolate B2-5 displayed an enhanced inhibitory activity against the pathogen mycelial growth with a temperature increase to 25°C, produced no pectinase (related to root rotting) and no critical rot symptoms at low [10(6) colony-forming units (CFU)/mL] and high (10(8) CFU/mL) inoculum concentrations. In pot experiments, pretreatment with the bacterial isolate in the presumed optimal time for disease control reduced disease severity significantly with a higher control efficacy at an inoculum concentration of 10(6) CFU/mL than at 10(8) CFU/mL. The establishment and colonization ability of the bacterial isolates on the ginseng rhizosphere appeared to be higher when both the bacterial isolate and the pathogen were coinoculated than when the bacterial isolate was inoculated alone, suggesting its target-oriented biocontrol activity against the pathogen. Scanning electron microscopy showed that the pathogen hyphae were twisted and shriveled by the bacterial treatment, which may be a symptom of direct damage by antifungal substances.

Conclusion: All of these results suggest that the bacterial isolate has good potential as a microbial agent for the biocontrol of the ginseng root rot caused by F. cf. incarnatum.

Keywords: Bacillus species; Fusarium cf. incarnatum; Panax ginseng Meyer; biological control; ginseng root rot.

Fig. 1

Phenotypic characteristics of Fusarium cf. incarnatum. (A) The cotton-like fungal colony formed on PDA. (B) An intercalary chlamydospore (Ch) in hypha (Hp). (C) Conidiophore (CP) and polyphialide (Ph) producing macroconidium (Mc). (D) Fusoid mesoconidia (Me), and macroconidia (Mc) slightly curved, frequently 3–5 septate, with a curved and tapering apical cell (Ac) and a foot-shaped basal cell (Bc). Bars, 20 μm.

Fig. 2

Rot symptom development on root discs. (A) Control (no rot). (B) Inoculated with 10⁴ conidia/mL (slight discoloration). (C) Inoculated with 10⁶ conidia/mL (severe rot) of the fungus after 6 d of incubation, and (D) on whole roots in the control (no rot). Inoculated with the fungus at inoculum concentrations of (E) 0.2% (mild rot symptoms), (F) 1%, and (G) 5% (severe rot symptoms) in the pot experiment.

Fig. 3

Mycelial growth of Fusarium cf. incarnatum after 7 d of incubation with bacterial isolates, showing that B2-5 and B8 had the strongest antifungal activity among the 12 bacterial isolates examined. Con: no treatment control. Bars and vertical lines are averages and standard deviations of three replications.

Fig. 4

Electron micrograph of the bacterial isolate B2-5, showing rod-shaped morphology with numerous peritrichous flagella. Bar, 1 μm.

Fig. 5

Effects of the bacterial isolate B2-5 on the inhibition of conidial germination (A) on solid medium and (B) in liquid medium, showing clear inhibition zones around the paper discs treated with (a) cell-free culture filtrate (Filtrate) and bacterial cell suspensions of (b) 10⁶ CFU/mL (10^6) and (c) 10⁸ CFU/mL (10^8), respectively, in (A), and conidial germination rates in bacterial treatments in (B). Marks and vertical lines in (B) are averages and standard deviations of three replications. Con, control.

Fig. 6

Effects of the bacterial isolate B2-5 with different inoculum concentrations [0 (Con), 10⁶ CFU/mL (10ˆ6)] and temperatures on rot symptom development on ginseng root discs, for which the degrees of rotting are 0, no rot; 1, 1–10%; 2, 10–30%; 3, 30–50%; 4, 50–70%; and 5, >70% (or fully) rotted. Mild rot symptoms (arrows) developed only at a high inoculum concentration (10⁸ CFU/mL) and at 25°C and 28°C. Arrowheads denote nonspecific decay by wounding.

Fig. 7

Effects of treatment times (pre-, simultaneous, and post-treatment) of the bacterial isolate B2-5 with different inoculum concentrations [0 (Con), 10⁶ CFU/mL (10⁶), and 10⁸ CFU/mL (10⁸)] on the inhibition of rot symptom development on ginseng root discs inoculated with Fusarium cf. incarnatum at 5 d after inoculation. Bars and vertical lines are means and standard deviations of nine replications. * Figures are inhibition rates (%) of rot symptom development relative to the control at the same treatment time. The same letters above the bars denote no significant difference within the same treatment time at p < 0.05 by the least significant different (LSD) test.

Fig. 8

Effects of bacterial treatment with inoculum on rot symptom development in ginseng roots caused by Fusarium cf. incarnatum at 10 d after pathogen inoculation. (A) Pathogen inoculation only with no bacterial treatment. Concentrations of (B) 10⁶ CFU/mL and (C) 10⁸ CFU/mL. (D) no bacterial treatment and pathogen inoculation.

Fig. 9

Population changes of the bacterial isolate B2-5 from low (L-; 10⁶ CFU/mL) and high (H-; 10⁸ CFU/mL) initial inoculum on ginseng root discs with (-F) and without (-N) inoculation of Fusarium cf. incarnatum. Marks and vertical lines are averages and standard deviations of nine replications.

Fig. 10

Scanning electron micrographs of Fusarium cf. incarnatum with no bacterial treatment (A), and treated with the bacterial isolate B2-5 at inoculum concentrations of (B) 10⁶ CFU/mL and (C) 10⁸ CFU/mL, showing wrinkled, distorted, and shrunken pathogen hyphae (Hy) adhered with bacterial cells (Ba), compared to intact hyphae (Hy) with smooth surface showing a contour of septum (St) in (A) the untreated control. Bars, 20 μm.