Anti-Oomycete Activity and Pepper Root Colonization of Pseudomonas plecoglossicida YJR13 and Pseudomonas putida YJR92 against Phytophthora capsici

Abstract

Previously, Pseudomonas plecoglossicida YJR13 and Pseudomonas putida YJR92 from a sequential screening procedure were proven to effectively control Phytophthora blight caused by Phytophthora capsici. In this study, we further investigated the anti-oomycete activities of these strains against mycelial growth, zoospore germination, and germ tube elongation of P. capsici. We also investigated root colonization ability of the bacterial strains in square dishes, including cell motility (swimming and swarming motilities) and biofilm formation. Both strains significantly inhibited mycelial growth in liquid and solid V8 juice media and M9 minimal media, zoospore germination, and germ tube elongation compared with Bacillus vallismortis EXTN-1 (positive biocontrol strain), Sphingomonas aquatilis KU408 (negative biocontrol strain), and MgSO4 solution (untreated control). In diluted (nutrient-deficient) V8 juice broth, the tested strain populations were maintained at >108 cells/ml, simultaneously providing mycelial inhibitory activity. Additionally, these strains colonized pepper roots at a 106 cells/ml concentration for 7 days. The root colonization of the strains was supported by strong swimming and swarming activities, biofilm formation, and chemotactic activity towards exudate components (amino acids, organic acids, and sugars) of pepper roots. Collectively, these results suggest that strains YJR13 and YJR92 can effectively suppress Phytophthora blight of pepper through direct anti-oomycete activities against mycelial growth, zoospore germination and germ tube elongation. Bacterial colonization of pepper roots may be mediated by cell motility and biofilm formation together with chemotaxis to root exudates.

Keywords: Phytophthora capsici; Pseudomonas plecoglossicida; Pseudomonas putida; bacterial colonization; pepper.

Fig. 1

Biocontrol activity of Pseudomonas plecoglossicida YJR13 and Pseudomonas putida YJR92 compared with Sphingomonas aquatilis KU408 (negative bacterial control) and Bacillus vallismortis EXTN-1 (positive bacterial control) 14 days after inoculation with Phytophthora capsici on 5-week-old pepper (cv. Nockwang) plants. Ten-mM MgSO₄ solution was used as an untreated control. Different letters above error bars (mean + standard error, n = 20) indicate significant (P < 0.05) differences between treatments according to the least significant difference test.

Fig. 2

Anti-oomycete activity of Pseudomonas plecoglossicida YJR13 and Pseudomonas putida YJR92 compared with Sphingomonas aquatilis KU408 (negative bacterial control) and Bacillus vallismortis EXTN-1 (positive bacterial control) against Phytophthora capsici mycelial growth on (A) V₈ juice agar and (B) M9 agar. Photographs were captured when mycelia in 10-mM MgSO₄ solution plates (untreated control) reached the center of the plates. Bacterial strains or MgSO₄ solution were streaked on the centers of the media 48 h before inoculation with P. capsici.

Fig. 3

Cell density-dependent inhibitory activity of Pseudomonas plecoglossicida YJR13 and Pseudomonas putida YJR92 compared with Sphingomonas aquatilis KU408 (negative bacterial control) and Bacillus vallismortis EXTN-1 (positive bacterial control) on V₈ juice agar. Five microliters of various concentrations of cell suspensions (10⁰ [10-mM MgSO₄ solution] to 10¹⁰ cells/ml) were applied on sterile filter paper disks. Mycelial plugs of the 5-day-old cultures of Phytophthora capsici were placed in the center of V₈ juice agar and incubated at 28°C in the dark until mycelia reached until mycelia covered the 10⁰ cell-treated paper disks.

Fig. 4

(A) Inhibitory activity of Pseudomonas plecoglossicida YJR13 and Pseudomonas putida YJR92 compared with Sphingomonas aquatilis KU408 (negative bacterial control) and Bacillus vallismortis EXTN-1 (positive bacterial control) against Phytophthora capsici mycelial growth 5 days after inoculation in diluted (0, 1/10, 1/50, 1/100, and 1/200) V₈ juice broth. (B) Bacterial populations (log colony-forming units [CFU]/ml) of P. plecoglossicida YJR13, P. putida YJR92, S. aquatilis KU408, and B. vallismortis EXTN-1 in the diluted V₈ broth 5 days after inoculation. Ten-mM MgSO₄ solution was used as an untreated control. Different lowercase and uppercase letters indicate significant (P < 0.05) differences between the dilutions in each treatment and between treatments according to the least significant difference test, respectively.

Fig. 5

(A) Swimming and (B) swarming activities of Pseudomonas plecoglossicida YJR13 and Pseudomonas putida YJR92 compared with Sphingomonas aquatilis KU408 (negative bacterial control) and Bacillus vallismortis EXTN-1 (positive bacterial control) 48 h after incubation on M9 minimal medium containing 0.3 and 0.5% (w/v) agar, respectively. These media were amended with 10 and 1,000 μM of (a) amino acids (aspartic acid, glutamic acid, glycine, and threonine), (b) organic acids (citric acid, fumaric acid, oxalic acid, and succinic acid), and (c) sugars (arabinose, fructose, glucose, and maltose), respectively. Different lowercase and uppercase letters on the columns (mean + standard error, n = 6) indicate significant differences between bacterial treatments at 10 and 1,000 μM concentrations at P < 0.05, respectively.