Cyclic lipopeptide profile of the plant-beneficial endophytic bacterium Bacillus subtilis HC8

Natalia Malfanova,Ben Lugtenberg,Marc Ongena,Laurent Franzil,Vladimir Chebotar

doi:10.1007/s00203-012-0823-0

Abstract

In a previous study (Malfanova et al. in Microbial Biotech 4:523–532, 2011), we described the isolation and partial characterization of the biocontrol endophytic bacterium B. subtilis HC8. Using thin-layer chromatography, we have detected several bioactive antifungal compounds in the methanolic extract from the acid-precipitated supernatant of HC8. In the present study, we have further analyzed this methanolic extract using liquid chromatography-mass spectrometry. Based on the comparison of retention times and molecular masses with those of known antifungal compounds, we identified three families of lipopeptide antibiotics. These include four iturins A having fatty acyl chain lengths of C14 to C17, eight fengycins A (from C14 to C18 and from C15 to C17 containing a double bond in the acyl chain), four fengycins B (C15 to C18), and five surfactins (C12 to C16). Evaluation of the antifungal activity of the isolated lipopeptides showed that fengycins are the most active ones. To our knowledge, this is the first report of an endophytic Bacillus subtilis producing all three major families of lipopeptide antibiotics containing a very heterogeneous mixture of homologues. The questions remain open which of these lipopeptides (1) are being produced during interaction with the plant and (2) are contributing to the biocontrol activity of HC8.

Highlights

Endophytes are plant-associated microbes that are able to colonize plants internally
LC–MS was performed on a crude methanolic extract of the acid-precipitated supernatant fluid of B. subtilis HC8
To elute all putative cyclic lipopeptides (c-LPs), we used a general elution program (Table 1a). This program uses a gradient of increasing amounts of acetonitrile and the first eluents include the less polar iturins followed by the increasingly polar fengycins and surfactins (Fig. 1)

Summary

Introduction

Endophytes are plant-associated microbes that are able to colonize plants internally. Due to the nature of their endophytic lifestyle, they establish a long-lasting stable relationship with a plant. In this symbiotic association, the plant provides nutrients and shelter for the microbes and, in turn, the endophyte can help the plant by protecting it against phytopathogens or by promoting its growth. One of the mechanisms of such a protection includes production of bioactive secondary metabolites which either can be directly involved in antibiosis Bacillus spp. are known to produce a wide range of secondary metabolites including cyclic lipopeptides (c-LPs), some of the most powerful ones with regard to their antifungal and biosurfactant activity (Ongena and Jacques 2008; Jacques 2011).

Objectives

Methods

Results