Suppressive potential of Paenibacillus strains isolated from the tomato phyllosphere against fusarium crown and root rot of tomato

Abstract

The suppressive potentials of Bacillus and Paenibacillus strains isolated from the tomato phyllosphere were investigated to obtain new biocontrol candidates against Fusarium crown and root rot of tomato. The suppressive activities of 20 bacterial strains belonging to these genera were examined using seedlings and potted tomato plants, and two Paenibacillus strains (12HD2 and 42NP7) were selected as biocontrol candidates against the disease. These two strains suppressed the disease in the field experiment. Scanning electron microscopy revealed that the treated bacterial cells colonized the root surface, and when the roots of the seedlings were treated with strain 42NP7 cells, the cell population was maintained on the roots for at least for 4 weeks. Although the bacterial strains had no direct antifungal activity against the causal pathogen in vitro, an increase was observed in the antifungal activities of acetone extracts from tomato roots treated with the cells of both bacterial strains. Furthermore, RT-PCR analysis verified that the expression of defense-related genes was induced in both the roots and leaves of seedlings treated with the bacterial cells. Thus, the root-colonized cells of the two Paenibacillus strains were considered to induce resistance in tomato plants, which resulted in the suppression of the disease.

Fig. 1

Suppressive effects against Fusarium crown and root rot by Paenibacillus strains 12HD2 and 42NP7 in a potted plant experiment. (A) Disease severity caused by Fusarium oxysporum f. sp. radicis-lycopersici in the roots of tomato seedlings treated with each strain. Each value indicates the mean of three experiments and the bar denotes the standard error of the mean. Different letters within each column indicate significant differences (P<0.05) according to Fisher’s LSD test. (B) Representatives of the washed roots of seedlings treated with SDW (left) and a cell suspension of 12HD2 (right). Note that rot symptoms were suppressed by the treatment with a cell suspension of 12HD2. (C) Density (CFU g⁻¹ of root) of Fusarium oxysporum isolated from the roots of potted tomato seedlings treated with each strain. The fungal species were also isolated from the healthy roots of uninoculated seedlings as a negative control (Healthy roots). Each value indicates the mean of three experiments and the bar denotes the standard error of the mean. Different letters within each column indicate significant differences (P<0.05) according to Fisher’s LSD test.

Fig. 2

Suppressive effects against Fusarium crown and root rot by Paenibacillus strains 12HD2 and 42NP7 in a field experiment. Each value indicates the mean of six experiments and the bar denotes the standard error of the mean. Different letters within each column indicate significant differences (P<0.05) according to Fisher’s LSD test.

Fig. 3

Disease severity caused by Fusarium oxysporum f. sp. radicis-lycopersici in the roots of potted tomato seedlings treated with bacterial cells and a culture filtrate of Paenibacillus strains 12HD2 and 42NP7. Each value indicates the mean of three experiments and bars denote the standard error of the mean. Different letters within each column indicate significant differences (P<0.05) according to Fisher’s LSD test.

Fig. 4

In vitro antifungal activities of the Paenibacillus strains 12HD2 and 42NP7 against Fusarium oxysporum f. sp. radicis-lycopersici on PDA (A) and 1.5% agar-supplied R2A (B) plates. Paper discs immersed with each cell suspension of strains 12HD2, 42NP7, Escherichia coli DH5α (negative control), and iturin-producing Bacillus amyloliquefaciens RC-2 (positive control) were placed on each agar plate containing budcells of Fusarium oxysporum f. sp. radicis-lycopersici. Observations were made 3 (A) and 10 (B) d after the incubation at 28°C. Note only the positive control formed clear zones, which indicated antifungal activity in both plates.

Fig. 5

Scanning electron microscopic images of the roots of semi-aseptic tomato seedlings treated with the cells of Paenibacillus strains 12HD2 (A, B) and 42NP7 (C, D). The circles in A–D indicate assemblages or clusters of cells at the interspaces between epidermal cells.

Fig. 6

Population dynamics of 42NP7rk in the roots of the tomato after the inoculation. The cell suspension of strain 42NP7rk, the antibiotic-resistant strain of 42NP7, was inoculated into the roots of tomato seedlings and the CFU was determined. Each value indicates the mean of three experiments and bars denote the standard error of the mean.

Fig. 7

Antifungal activity in acetone extracts from the roots of tomato seedlings treated with bacterial cells of Paenibacillus strains 12HD2 and 42NP7 against FORL (A) and an indicator phytopathogenic fungus, Colletotrichum dematium (B). Antifungal activity was evaluated based on the relative diameter of mycelial growth from a small mycelial block of the fungus relative to growth following a treatment with a control solution (50% methanol). Each value indicates the mean of three experiments and bars denote the standard error of the mean. Different letters within each column indicate significant differences according to Fisher’s LSD test.

Fig. 8

Expression of defense-related genes in the leaves and roots of tomato seedlings treated with the cells of Paenibacillus strains 12HD2 and 42NP7. SA-responsive (PR-1, PR-5) and JA/ET-responsive (PR-3, PR-6) genes were used as representative markers in RT-PCR. The expression of genes in the seedlings treated with Escherichia coli DH5α are displayed as a control. The actin gene, Act, was used as an internal standard for expression. Act (not RT) represents the amplification of Act using nonreverse-transcripted RNA as a negative control.