Isolation and antifungal and antioomycete activities of aerugine produced by Pseudomonas fluorescens strain MM-B16

Abstract

The bacterial strain MM-B16, which showed strong antifungal and antioomycete activity against some plant pathogens, was isolated from a mountain forest soil in Korea. Based on the physiological and biochemical characteristics and 16S ribosomal DNA sequence analysis, the bacterial strain MM-B16 was identical to Pseudomonas fluorescens. An antibiotic active against Colletotrichum orbiculare and Phytophthora capsici in vitro and in vivo was isolated from the culture filtrates of P. fluorescens strain MM-B16 using various chromatographic procedures. The molecular formula of the antibiotic was deduced to be C(10)H(11)NO(2)S (M(+), m/z 209.0513) by analysis of electron impact mass spectral data. Based on the nuclear magnetic resonance and infrared spectral data, the antibiotic was confirmed to have the structure of a thiazoline derivative, aerugine [4-hydroxymethyl-2-(2-hydroxyphenyl)-2-thiazoline]. C. orbiculare, P. capsici, and Pythium ultimum were most sensitive to aerugine (MICs for these organisms were approximately 10 micro g ml(-1)). However, no antimicrobial activity was found against yeasts and bacteria even at concentrations of more than 100 micro g ml(-1). Treatment with aerugine exhibited a significantly high protective activity against development of phytophthora disease on pepper and anthracnose on cucumber. However, the control efficacy of aerugine against the diseases was in general somewhat less than that of the commercial fungicides metalaxyl and chlorothalonil. This is the first study to isolate aerugine from P. fluorescens and demonstrate its in vitro and in vivo antifungal and antioomycete activities against C. orbiculare and P. capsici.

FIG. 1.

Transmission electron micrograph of bacterial strain MM-B16 grown on tryptic soy agar for 24 h. Bar = 1 μm.

FIG. 2.

Phylogenetic location of bacterial strain MM-B16 based on 16S rDNA sequences. The phylogenetic tree was constructed based on the percent difference in the genetic relationships between the allied strains. The length of each pair of branches represents the distance between sequence pairs, while the units at the bottom of the tree indicate the number of substitution events.

FIG. 3.

Sephadex LH-20 column chromatogram of active fractions from the eluates of preparative TLC. The Sephadex LH-20 column (26 by 950 mm) was eluted with methanol at a flow rate of 0.15 ml min⁻¹. All fractions were bioassayed for the antifungal and antioomycete activity against C. orbiculare and P. capsici using the paper disk-agar diffusion method.

FIG. 4.

EI mass spectrum of the antibiotic aerugine.

FIG. 5.

FT-IR spectrum of the antibiotic aerugine.

FIG. 6.

(A). Correlations of partial structures of the antibiotic aerugine from HMBC spectra; (B) structure of the antibiotic aerugine isolated from P. fluorescens strain MM-B16.

FIG. 7.

In vivo efficacy of aerugine for control of plant diseases in host plants. (A) Effects of aerugine and metalaxyl on disease development in pepper plants inoculated with P. capsici at the first-branch stage. Disease severity is based on a scale from 0 to 5, where 0 indicates no visible symptoms and 5 indicates a dead plant. (B) Effects of aerugine and chlorothalonil on anthracnose development on cucumber leaves inoculated with C. orbiculare. Numbers of lesions on the leaves of cucumber plants were rated on day 6 after inoculation. Means at each concentration labeled with the same letter are not significantly different (P = 0.05) according to the least significant difference test.