Purification and structural characterization of fengycin homologues
produced by Bacillus subtilis from poplar wood bark
Received: 6 September 2017 /Accepted: 5 March 2018 /Published online: 23 March 2018
Australasian Plant Pathology Society Inc. 2018
Poplar fungi canker is a disease that constitutes a serious threat to the poplar plant, a model tree in forest research. The control of
this disease by use of chemical fumigants is associated with unwanted side effects such as environmental pollution and drug
resistance. However, poplar canker can be effectively controlled with fengycin, an environmentally friendly bio-surfactant of
microbial origin. Fengycin homologues are a series of lipopeptides with variations in both the length and branching of the β-
hydroxy fatty acid. This study was aimed at purifying and characterizing fengycin homologues produced by Bacillus subtilis
from poplar wood bark. A total of 13 fengycin homologues were obtained through 70% ammonium sulfate precipitation,
methanol extraction, ion exchange chromatography, gel filtration chromatography, and semi-preparative high-performance liquid
chromatography (HPLC). Electro-spray ionization mass spectrometry (ESI-MS) and electro-spray ionization tandem mass
spectrometry (ESI-MS/MS) were employed to elucidate the chemical structures of these compounds, and the structures were
further characterized by amino acid analysis. Thirteen (13) of the active compounds were fengycin A or B homologues. All the 13
homologues exhibited antifungal activity against the indicator strain Botryosphaeria dothidea. This finding is considered sig-
nificant, in that it is the first report on the production of so many fengycin homologues and isomers by Bacillus subtilis. Thus,
B. subtilis from poplar wood bark is a rich source of environment-friendly, anti-microbial agents for control of poplar canker.
Keywords Bacillus subtilis
Poplar is a model tree in forest research (Bradshaw et al.
2000). However, the development of poplar plantation in
China is hampered by poplar canker, a disease that causes
local necrosis in poplar branches. Poplar canker comprises
two types: poplar bacteria canker and poplar fungi canker
(Ma et al. 2015). Poplar fungi canker is caused by some path-
ogenic fungi, such as Botryosphaeria dothidea (Jiang et al.
2015), and Cytospora chrysosperma (Jia et al. 2010).
Although chemical treatments are useful in protecting poplar
plants from the disease, overuse of chemical fungicides can
cause environmental pollution and increased drug resistance.
One promising and environmentally-friendly alternative
method involves biological control, which uses microorgan-
isms to kill or inhibit pathogenic organisms (Cazorla and
Bio-surfactants are microbial metabolites with good emul-
sifying and surface activities (Coronel-León et al. 2015).
Lipopeptides are the most important bio-surfactants in envi-
ronmental applications (Ghojavand et al. 2011). Lipopeptides
include iturin, surfactin and fengycin (Rautela et al. 2014;Ma
et al. 2015). Antimicrobial lipopeptides and their homologues
or isomers are a group of active peptides with similar struc-
tures or identical molecular weight (Pathak et al. 2014).
Fengycin, one of the most important and promising
lipopeptides, is produced by Bacillus species (Ongena et al.
2005). Fengycin is a promising antifungal agent against path-
ogenic fungi (Wang et al. 2004). It exists as a mixture of
homologues or isoforms which differ in fatty acid (FA) chain
lengths, and in the amino acid composition of the peptide ring
(Vanittanakom et al. 1986). Fengycin homologues include
* Xunli Liu
College of Life Sciences, Shandong Agricultural University,
Tai’an 271018, China
School of Life Sciences, Taishan Medical University, Tai’an 271018,
College of Forestry, Shandong Agricultural University,
Tai’an 271018, China
Australasian Plant Pathology (2018) 47:259–268