An Endophytic Sanguinarine-Producing Fungus from Macleayacordata, Fusarium proliferatum BLH51

Xue-Jun Wang • Chang-Li Min • Mei Ge •

Rui-Hua Zuo

Received: 4 September 2013 / Accepted: 11 September 2013 / Published online: 29 October 2013

� Springer Science+Business Media New York 2013

Abstract Fermentation processes using sanguinarine-

producing fungi other than Macleaya cordata may be an

alternative way to produce sanguinarine (SA), which is a

quaternary benzo[c]phenanthridine alkaloid possessing

antibacterial, anthelmintic, and anti-inflammatory proper-

ties. In this study, a SA-producing endophytic fungus strain

BLH51 was isolated from the leaves of M. cordata grown

in the Dabie Mountain, China. Strain BLH51 produced SA

when grown in potato dextrose liquid medium. The amount

of SA produced by this endophytic fungus was quantified

to be 178 lg/L by HPLC, substantially lower than that

produced by the host tissue. The fungal SA—which was

analyzed by thin layer chromatography and high-perfor-

mance liquid chromatography—was shown to be identical

to authentic SA. Strain BLH51 was identified as Fusarium

proliferatum based on the morphological characteristics

and nuclear ribosomal DNA ITS sequence analysis. To the

best of our knowledge, this is the first report concerning the

isolation and identification of endophytic SA-producing

fungi from the host plant, which further proved that

endophytic fungi are valuable reservoirs of bioactive

compounds.

Introduction

Macleaya cordata (Willd) R. Br., also known as plume

poppy or Bocconia cordata is a perennial plant of the Pap-

averaceae family. It has been used as one of traditional

Chinese medicines for a long time. This herb widely dis-

tributed throughout southeastern and northwest of China.

Sanguinarine (SA), a quaternary benzo[c] phenanthridine

alkaloid (QBA), isolated from M. cordata, Sanguinaria

canadensis, and Chelidonium majus are known to exert a

wide spectrum of biological activities, such as antibacterial

[1, 2], anthelmintic [3], antitumour [4, 5], and anti-inflam-

matory properties [6, 7]. QBA fractions from M. cordata

(SANGUIRITRIN) and S. canadensis (SANGUINARIA)

are used in toothpastes and mouthwashes as antiplaque

agents. SANGUIRITRIN is applied as a veterinary prepa-

ration for mastoiditis in cows [8]. SA is an active component

of the preparation Sangrovit� as an additive to animal feeds.

Recently, SA has gained increasing attention as potential

agents in the treatment of cancer.

Most of the SA currently used in herbal supplements and

medicines is extracted from M. cordata and some other

species in the Papaveracea. Although M. cordata actually

has abundant SA content, and it has a rich resources, but

indiscriminate felling of trees will destroy the ecological

balance of nature. Thus, increasing efforts have been made

to develop alternative means of SA production, such as

using complete chemical synthesis and Papaveracea plant

cell culture. However, thus far, the total chemical synthesis

of SA still cannot be chemically synthesized in an indus-

trially feasible manner, and the in vitro culture of opium

poppy has only met limited success [9].

In recent years, endophytic fungi from plants have been

widely accepted as major sources of drugs, and a large

number of compounds with new structures and various

X.-J. Wang � C.-L. Min (&) � R.-H. Zuo

College of Biotechnology and Pharmaceutical Engineering,

West Anhui University, Lu’an 237012, China

e-mail: mcl0917@163.com

X.-J. Wang � C.-L. Min

Research Center for Endophytic Fungi Resources of Dabie

Mountain, West Anhui University, Lu’an 237012, China

M. Ge

Shanghai Laiyi Center of Biopharmaceutical R&D,

Shanghai 201203, China

123

Curr Microbiol (2014) 68:336–341

DOI 10.1007/s00284-013-0482-7

bioactivities are continuously being isolated from them [10].

Thus, if a microbial source of SA is available, there is no need

to harvest and extract the M. cordata for this drug. As far as

we know, little work has been done concerning the endo-

phytic fungi associated with M. cordata. Therefore, this

study aimed to isolate the endophytic SA-producing fungus

in M. cordata plants collected from Anhui Province of China

by rDNA ITS sequences analysis, and the potential of strain

BLH51 for SA production was also evaluated.

Materials and Methods

Materials

Solvents used for chromatography were of high-perfor-

mance liquid chromatography (HPLC) grade, while sol-

vents used for extraction were of American Chemical

Society grade. Authentic SA (C98 % purity) was obtained

from the National Institute for the Control of Pharmaceu-

tical and Biological Products of China. All other chemicals

were purchased from China Medicine Shanghai Chemical

Reagent Co., Ltd. Polymerase chain reaction (PCR) prim-

ers were synthesized by Shanghai Sangon Biologic Engi-

neering Technology and Service Co., Ltd.

Samples of M. cordata were obtained from the natural

populations at Dabie Mountain in Anhui Province in cen-

tral China. All samples were placed in polyethylene bags,

immediately transported to the laboratory, and placed in a

refrigerator at 4 �C.

Isolation of Endophytic Fungi from Macleaya cordata

Strain BLH51 used in this study was 1 of 55 endophytic fungi

isolated from the leaves of M. cordata. Healthy leaves of

M. cordata were thoroughly washed in running tap water, then

sterilized by washing in 70 % ethanol for 2 min and 0.5 %

sodium hypochlorite for 2 min, and followed by washing with

70 % ethanol (v/v) for 5 s. Afterwards, the leaves were rinsed

four times in sterile distilled water, and then they were cut into

pieces (0.5 9 0.5 cm). The small pieces were placed in petri

dishes (9 cm diameter) on the surface of potato dextrose agar

(PDA) medium containing 0.5 g/L streptomycin sulfate. Petri

dishes were incubated at 28 �C in darkness. After several days,

fungi were observed growing from the leaf fragments. Indi-

vidual hyphal tips of the various fungi were removed and

placed on fresh PDA medium and incubated at 28 �C for at

least 10 days. Each fungal culture was checked for purity and

transferred to another PDA plate by the hyphal tip method. The

fungal isolates were numbered and stored on the surface of

PDA plate at 4 �C or as spores and mycelia in 15 % (v/v)

glycerol at -70 �C.

Fermentation and Preparation of Endophytic Fungi

Extracts

The endophytic fungi isolates were inoculated, respectively,

into 500 mL Erlenmeyer flasks containing 100 mL of potato

dextrose liquid medium and cultured at 28 �C with 200

recycles/minute for 10 days in a rotary shaker. The mycelia

were harvested by centrifugation at 12,0009g for 10 min and

dried at 50 �C overnight. Dried mycelia were crushed and

extracted with 85 % ethanol under reflux for 1 h at 80 �C,

repeated four times, the extracts were filtered and the filtrates

were evaporated under reduced pressure. The dry residue was

dissolved in 50 mL 1 % sulfuric acid–water, and then

exhaustively extracted with chloroform. In the last stage, the

combined chloroform extracts were evaporated to dryness.

The dry residues were dissolved in 1 mL of methanol (HPLC

purity grade). The methanolic extracts were filtered through a

0.45 lm filter prior to chromatographic separation.

Screening of SA-Producing Endophytic Fungi

The SA-producing endophytic fungi were screened by thin

layer chromatography (TLC). TLC analysis of the metha-

nolic extract of endophytic fungi as well as the methanolic

solution of SA standard was developed in a solvent system

(petroleum ether: methanol: at 15:1 v/v) by spotting on a

0.25 mm (10 9 20 cm) silica gel plate. SA was detected

under 365 nm ultraviolet light, which appeared as yel-

lowish spots. The SA spot was identified by comigration

with authentic SA.

High-performance liquid chromatography (HPLC) was

performed using a C18 column (5 lm, 4.6 9 150 mm)

(Agilent, USA). A 20 lL amount of each methanolic

extract was injected. The mobile phase was acetonitrile:

water (25: 75, v/v) at a flow rate of 1.0 mL/min. The

effluent was monitored at 270 nm. SA was quantified by

comparing the peak area of the samples with that of the

authentic SA.

Identification of Strain BLH51

After SA was determined in the culture of strain BLH51, it

was identified by microscopic morphologic characteristics

and ITS sequence analysis. The strain BLH51 was grown

on the surface of PDA medium at 25 �C for 2 weeks,

followed by identification based on the morphology of the

fungal colony, and the characteristics of the spores.

Mycelia and conidia of strain BLH51 were observed with a

light microscope (BA300, Motic, China).

The genomic DNA of strain BLH51 was extracted from

fresh mycelia according to the method described by Pirttila

X.-J. Wang et al.: Producing Fungus from Macleaya cordata 337

123

et al. [11]. The target rDNA region including ITS1, ITS2

regions and 5.8S gene was amplified using primers ITS1 (50-TCCGTAGGTGAACCTGCGG-30) and ITS4 (50-TCCTCC

GCTTATTGATATGC-30) [12]. Total volume was brought

to 25 lL with deionised water. Amplification of the ITS

region was performed as follows: 95 �C for 3 min, followed

by 40 cycles of 94 �C for 60 s, 50 �C for 60 s, and 72 �C for

120 s and a final extension at 72 �C for 10 min. The PCR

products were purified using the Gel extraction kit (Watson,

China) and sequenced by Shanghai Sangon Biologic Engi-

neering Technology and Service Co., Ltd.

The ITS sequence of strain BLH51 was compared to the

data available in NCBI using BLASTn search to estimate

the phylogenetic relationships of the endophytic fungi. The

resulting sequences were aligned with the Clustal X soft-

ware [13] with gaps treated as missing data. The phylo-

genetic tree was constructed using the neighbor-joining

method [14] and the Kimura two-parameter distance cal-

culation in mega software version 3.1 [15]. The bootstrap

was 1,000 replications to assess the reliable level to the

nods of the tree.

Results

Isolation of Endophytic Fungi

We obtained 55 endophytic fungi isolates flourishing in the

leaves of M. cordata from Dabie Mountain, China. Based

on colonial characteristics, the endophytic fungi were

related to Fusarium spp., Aspergillus spp., Penicillium

spp., Leptosphaeria spp., and unidentified strains. These

results confirm host specificity and geographic structure

affect biological diversity of endophytic fungi [16, 17].

Screening of SA-Producing Endophytic Fungi

The extracts of fungal cultures were examined for the

presence of SA by TLC under UV illumination. Results

indicated that strain BLH51 showed positive results for SA

production in potato dextrose liquid medium, and one of

the fungal compounds exhibited the same Rf values (0.69)

as authentic SA (Fig. 1).

Results of HPLC analysis also confirmed the presence of

SA by showing a retention time of 12.111 min, which was

similar to authentic SA (12.082 min) (Fig. 2). The SA

Fig. 1 Thin layer chromatogra-

phy analysis of authentic san-

guinarine (a), and fungal

BLH51 sanguinarine formation

in potato dextrose broth (b) on

silica gel. Arrow indicates the

presence of sanguinarine

AU

0.00

0.02

0.04

0.06

0.08

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

AU

0.00

0.02

0.04

0.06

0.08

Minutes2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00

A

B

Fig. 2 High-performance

liquid chromatogram of

authentic sanguinarine (a) and

fungal sanguinarine (b). The

mobile phase was acetonitrile/

water (25:75, v/v) with a flow

rate of 1.0 mL/min.

Registrations of peak and

retention time were recorded by

UV detection at 270 nm. Fungal

sample showed a peak with

retention time 12.111 min,

which was found to be identical

to authentic sanguinarine

338 X.-J. Wang et al.: Producing Fungus from Macleaya cordata

123

yield of strain BLH51 was about 178 lg/L or 135 lg/g (SA

per dry wt of mycelium) when it was cultured in 100 mL

potato dextrose liquid medium at 28 �C with 200 recycles/

minute shaking for 10 days. Our results showed the fungal

endophyte clearly produced SA.

Phenotypic Characterization of Strain BLH51

The endophytic fungus, BLH51, was cultivated on PDA at

25 �C for 14 days. The colonial morphological traits of the

BLH51 isolate were white, then lilac tinge in the central of

colony, cottony, and nearly round margins (Fig. 3a, b).

Macroconidia were slender, almost straight to slightly curved,

and the size of macroconidia averaged 20–48 9 2.1–3.7 lm.

Microconidia were pyriform, club-shaped, measuring

4–11 9 2–3 lm, on average (Fig. 3c). The morphological

characteristics of strain BLH51 are similar to those of

Fusarium. proliferatum described by Nirenberg and

O’Donnell [18]. Based on the results of morphological tests,

strain BLH51 was identified as F. proliferatum.

ITS rDNA Sequence and Phylogenetic Analysis

To further determine the genetic relationships of strain

BLH51, the ITS fragments were amplified and sequenced,

including 534 bp (GenBank accession number KC461140).

After homology searching against GenBank, the sequence

was found to share 100 % similarity with F. proliferatum

(GenBank accession number HQ380763). A phylogenetic

relationship was established through alignment and cla-

distic analysis of homologous nucleotide sequences among

these fungal species (Fig. 4). Strain BLH51 was shown to

be closest to the genus Fusarium. So the isolate was further

identified as F. proliferatum.

Fig. 3 Morphological

observation of F. proliferatum

BLH51. a The colonies after

5 days at 25 �C on PDA,

b Reverse side of the colony;

c The morphological

characteristics of hyphae and

conidiophores by light

microscope (4009)

Fusarium napiforme(HQ165918)

Gibberella moniliformis(DQ655723)

Fusarium guttiforme(GU205427)

Fusarium austroamericanum(DQ459839)

Gibberella intricans(KC005676)

Fusarium camptoceras(EU520082)

BLH51(KC461140)

Fusarium proliferatum(AF291061)

Fusarium proliferatum(HQ607967)

Fusarium proliferatum(HQ380763)

Fusarium proliferatum(GQ265957)

Fusarium lateritium(DQ655721)

Fusarium avenaceum(KC354489)

Geejayessia cicatricum(HQ728145)

Albonectria albosuccinea(HQ897788)

Epichloe elymi(DQ899096)

74

99

98

97

97

100

60

58

66

58

0.02

Fig. 4 Phylogenetic tree of the

F. proliferatum, BLH51. The

tree was gave on the basis of

ITS sequences, using the

neighbor-joining method.

Numbers at nodes are bootstrap

scores (above 50 %) obtained

from 1,000 replications

X.-J. Wang et al.: Producing Fungus from Macleaya cordata 339

123

Discussion

This study isolated a SA-producing endophytic fungus

BLH51 from the leaves of M. cordata obtained from Dabie

Mountain in Anhui Province in central China. Strain BLH51

was identified as F. proliferatum on the basis of its mor-

phology and the ITS sequence. Previous studies demonstrated

that Fusarium spp. are distributed worldwide, and are fre-

quently obtained from some trees and numerous crop plants

[19–21]. The genus Fusarium includes many species, with F.

proliferatum as one of the most common species. Fusarium

spp. was reported to produce several secondary metabolites,

including cyclic tetrapeptides, rohitukine, taxol, and other

antibiotics [19, 21–23]. To the best of our knowledge, the

endophytic fungus Fusarium spp. and other genus have never

been reported capable of producing SA. The present study is

the first to isolate, characterize, and identify SA-producing F.

proliferatum from Papaveraceae plants in China.

The discovery of SA-producing endophytic fungi asso-

ciated with Papaveraceae plants is valuable for industrial

interest and for basic research. The consistent production of

SA by F. proliferatum BLH51 further supports the theory

that during the long coevolution of endophytes and their host

plants, endophytes adapted to their special microenviron-

ments by genetic variation, including uptake of some plant

DNA into their own genomes [24]. This could have led to the

ability of certain endophytes to biosynthesize some phyto-

chemicals originally associated with the host plant [25].

The total amount of SA produced by F. proliferatum

BLH51 was 178 lg/L or 135 lg/g (SA per dry wt of

mycelium) when it is cultured under the conditions described

in this study. This suggests that F. proliferatum BLH51 is a

promising candidate for SA production. However, it is only a

wild strain, and we believe that the SA yield of F. prolifer-

atum BLH51 will be increased by strain improvement and

optimization of the fermentation media.

Acknowledgments This work was supported by National Natural

Science Foundation of China (Grant No. 31100019) and Anhui Nat-

ural Science Research Project of Colleges and Universities of China

(Grant Nos. KJ2013A266 and KJ2011Z398). We thank Prof. Yun-

jiang Min from College of Biotechnology and Pharmaceutical Engi-

neering, West Anhui University, China for helping with the identifi-

cation of the plant material. We also wish to thank the anonymous

reviewers for constructive comments.

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