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Journal of Medicinal Plants Research Vol. 4(24), pp. 2633-2640, 18 December, 2010
Available online at http://www.academicjournals.org/JMPR
ISSN 1996-0875 ©2010 Academic Journals
Full Length Research Paper
Enhanced dragon’s blood production in Dracaena
cochinchinensis by elicitation of Fusarium oxysporum
strains
XingHong Wang1, Changhe Zhang2, LingLing Yang1, Xiao-Hong Yang3, Ji-Dong Lou4,
Qiu’e Cao5* and Jose Gomes-Laranjo2
1Yunnan Institute of Microbiology, Yunnan University, North Road of Green Lake 2, Kunming 650091, China.
2Centre for the Research and Technology of Agro-Environmental and Biological Sciences (CITAB)/Department of
Biology and Environment, Universidade de Trás-os-Montes e Alto Douro (UTAD), Apartado 1013,
5001-801 Vila Real, Portugal.
3College of Life Science and Technology, Kunming University, Kunshi Road 2, Kunming 650031, China.
4College of Life Sciences, China Institute of Metrology, Hangzhou 310018, China.
5College of Chemical and Material, Yunnan University, North Road of Green Lake 2, Kunming 650091, China.
Accepted 4 October, 2010
Dragon’s blood is a traditional medicine broadly used in the world for many centuries. The objective of
this work was to screen microorganisms to enhance and control its production. Twenty microbial
strains were isolated from the stem xylem of Dracaena cochinchinensis. Compared with wounding
alone, inoculation with living mycelia of isolates YM-2617 and YM-6113 on a fresh wound significantly
increased the dragon’s blood yield by 2.9- and 2.3-fold, respectively. The two strains were identified as
Fusarium oxysporum by morphology and 16S rDNA sequence analysis. The fungal induced dragon’s
blood had a similar chemical constituent to that of the natural dragon’s blood as analyzed by UPLC. In
addition, it had a similar or higher antimicrobial activity than that of the natural dragon’s blood. These
results indicate that elicitation by F. oxysporum has the potential to artificially control dragon’s blood
production in a sustainable way without destroying the valuable endangered trees.
Key words: Fusarium oxysporum, Dracaena cochinchinensis, antimicrobial activity, dragon’s blood production,
loureirin a and b.
INTRODUCTION
Dragon’s blood is a dark red resin, which has been one of
the most valuable traditional medicines extensively used
by many cultures for lots of centuries (Sousa et al.,
2008). It is originally obtained from the trees of Dracaena
spp. Because of the rare resources, resins from
Daemonorops (Palmae), Croton (Euphorbiaceae) and
Pterocarpus (Fabaceae) are used as alternatives (Gupta
et al., 2008). Dragon’s blood has antimicrobial,
*Corresponding author. E-mail: qecao@ynu.edu.cn. Tel: 0086-
8715033723. Fax: 0086- 8715033726.
antioxidant, anti-inflammatory, antitumor and cytotoxic
activities. It dispels blood stasis, stops bleeding and
promotes healing (Liu et al., 2005). Oral administration of
it could stimulate blood circulation and relieve pain,
mainly for the treatment of cardiovascular disorders such
as coronary heart disease, cerebral infarction, myocardial
and cerebral ischemia, Raynaud’s disease and other
thrombotic diseases; external application of it could stop
bleeding, promote wound healing, mainly used for
various skin or mucosal disease, such as diabetic foot
ulcers and pressure ulcers. Dracaena cochinchinensis
(Lour.) S. C. Chen is a recently discovered native
dracaena species that has become the main source of
2634 J. Med. Plant. Res.
dragon’s blood in China since 1970’s (Cai and Xu, 1979).
Pharmaceutical dragon’s blood is the dry alcohol extract
of the resinous wood of dracaena trees. The main
components of dragon’s blood are believed to be
flavonoids and stilbenoids (phenolics) (Fan et al., 2008)
biosynthesized through the phenylpropanoid pathway.
However, the major effective species of dragon’s blood
are still unclear. The content of loureirins a and b is
arbitrarily selected for the evaluation of the quality of
dragon’s blood in China. The formulas of these two
compounds are illustrated above.
D. cochinchinensis grows extremely slow with very low
dragon’s blood yield. There is no secretory tissue to
secrete dragon’s blood so it stays in its origin stem xylem
parenchyma cells (Fan et al., 2008). To harvest a few
pieces of resinous wood, a tree with hundreds of years
old is often destroyed. Owing to overexploitation, the two
native Dracaena species D. cochinchinensis and D.
cambodiana have been included in the third protection
group of China’s endangered species (Anon, 1987). The
current annual demand for dragon’s blood in China is
more than 600 tons, mainly depending on import.
However, according to International Union for the
Conservation of Nature and Natural Resources (IUCN),
other Dracaena spp. that produce dragon’s blood, such
as Dracaena draco and Dracaena cinnabari are also
endangered and has been presented in the IUCN red list
since 1998 (Banares, 1998) and 2004 (Miller, 2004),
respectively (www.iucnredlist.org).
Large-scale cultivation of dracaena trees might be the
only potential way to solve the dragon’s blood shortage
problem and to protect the nature. Nevertheless, the
production of dragon’s blood is uncertainty. At what time
and at which part of the trunk a tree will produce dragon’s
blood and how much will be produced are unclear. Little
is known about the formation mechanism on dragon’s
blood; there is no efficient method to promote or induce
dragon’s blood formation. The formation of agarwood
resin (chen-xiang, gaharu, jinko or aloeswood), which
also occurs in the stem or root xylem, is believed to be
the result of Aquilaria spp. response to fungal infection
(http://www.cropwatch.org/agarwood.htm).
Microorganisms especially fungi are often observed
near resinous part of the stem of D. cochinchinensis in
natural environment (Wang et al., not published data). We
thus supposed that microorganisms might be involved in
the formation of dragon’s blood. This work aimed to
screen microorganisms in order to artificially enhance
dragon’s blood production.
MATERIALS AND METHODS
Microbial isolation and inoculation
The microbial strains were isolated from the stem xylem of wild D.
cochinchinensis trees aged 50 - 100 years located in Simao and
Xishuangbanna, Yuannan Province. The trees with dragon’s blood
were selected for the isolation. The xylem parts next to the resinous
wood were cut off and disinfected with 70% alcohol for one minutes
The xylem was cut into 0.5 cm3 chips used as microbial resources
and subject to general screening procedure on sterile potato
dextrose agar (PDA) plates. The isolates were purified by single-
colony or single-spore method. All the purified strains were
activated at 25°C for one week for inoculation to the hole in stem
xylem. Each tested group contained 10 holes. The distance
between two neighboring holes was about 5 cm. Three to five test
groups and control groups with agar media but without any
microorganism were performed in each tree.
Harvesting and extraction of dragon’s blood
The produced dragon’s blood in the xylem in the inoculation site
was collected by removing the surrounding bark and digging up all
the red resinous wood. The resinous wood was cut into small
pieces and dried at 70°C for five h and extracted with five times of
methanol (V/W) for 3 times. The methanol extract was evaporated
with a vacuum rotary evaporator at 65°C to remove methanol. The
dry weight (dw) of the dragon’s blood was obtained then.
UPLC analysis of the dragon’s blood
The UPLC analysis was performed on a waters acquity ultra
performance LC system (Waters, USA) consisting of a waters
2996PDA detector and waters acquity UPLC BEH C18 column (2.1
× 50 mm, 1.7 m) using a mixture of acetonitrile and water (29:71,
v/v) as mobile phase at 0.3 ml/min. The column was covered with a
protector at 40°C. The injection volume was 5 l. The standard
natural dragon’s blood was purchased from Kunming Daguan
Pharmaceutical Company (Kunming, China). Loureirin a and b were
measured by standard addition methods. The standards of these
two compounds were from China National Institute for the Control of
Pharmaceutical and Biological Products (NICPBP). All other
compounds are from Sigma.
Anti-microbial tests
Seven microorganisms covering the common major types of
microorganism were used for the antimicrobial assay. The fungal
strains were tested in PDA media; bacterial strains were tested in
beef-extract peptone media. All of the 7 strains were preserved in
Yunnan Institute of Microbiology.
Overnight cultures of the bacteria and yeast were adjusted to 2 -
5 × 108 c.f.u./ml. One ml of such a culture was mixed with 15 ml of
medium with the dragon’s blood in Petri dishes (9 cm in diameter).
The 24 h cultures of the mycelium fungi were inoculated at three
points on each PDA plate. The cultures were incubated at 37°C for
16 - 24 h for the analysis of bacteria and at 28°C for 72 - 96 h for
the analysis of fungi.
Strain identification
Morphology observation
According to the method described by Leslie and Summerell
(2006), single spores were incubated on carnation leaf-piece agar
(CLA) and PDA plates at 25°C for character observation with a
microscope (Olympus BH-2 with a LY-WN-HP CCD, manufactured
by Chendu Liyang Precision Electromechanical Co., Ltd).
ITS sequence analysis
The genomic DNA were extracted as described by Boekhout et al.
(1995). The rDNA ITS gene were amplified by PCR using primers
ITS1 and ITS4 (Gardes and Bruns, 1993) by an initial denaturing
step of 5 min at 94°C, followed by 30 cycles of 30 s at 94°C, 30 s at
55°C and 60 s at 72°C, with a final extension step of 5 min at 72°C.
A preliminary sequence similarity search was performed against
known sequences available in the GenBank using Blast (Altschul et
al., 1997). Multiple alignments with corresponding nucleotide
sequences of representatives of the genus Fusarium retrieved from
GenBank were carried out using clustal x program (Thompson et
al., 1997). Positions where gaps existed in any of the aligned
sequences were excluded. The neighbour-joining (NJ) phylogenetic
tree was performed using the software package mega version 4
(Kumar et al., 2004), and evaluated using the bootstrap values
(Felsenstein, 1985) based on 1000 replicates.
RESULTS
Two fungal isolates enhanced dragon’s blood
production
Twenty microbial strains were isolated from the stem of
D. cochinchinensis. Among the 20 isolates, two strains
namely YM-2617 and YM-6113 induced the stem xylem
of D. cochinchinensis producing dragon’s blood. As
shown in Figure 1, wounding and keeping the wound
moist afterwards were essential for dragon’s blood
formation, that is an open wound dried quickly and did
not produce dragon’s blood (control 1). Compared with
control 2 (wounding and keeping the wound moist),
strains YM-2617 and YM-6113 significantly increased
dragon’s blood yield by 2.9- and 2.3-fold (P < 0.001),
respectively. Inoculation of autoclaved dead mycelia of
YM-2617 and YM-6113 significantly increased dragon’s
Wang et al. 2635
blood yield by 0.8- and 0.7-fold (P < 0.01), respectively.
This indicated that dragon’s blood was produced by the
plant xylem cells not by the fungal cells. Flourishing
fungal mycelia with spores were often observed in the
control holes, which maybe came from the environment;
however, only sparse mycelia without spores was found
in the holes inoculated with strain YM-2617 or YM-6113.
This might be because of the higher dragon’s blood
content in the fungal induced wood. Other 16 isolates did
not give significant difference in dragon’s blood yield
compared with control 2; another two strains had strong
pathogenesis resulting in anthracnose without dragon’s
blood production at the wounded area.
The age of the tree did not affect the dragon’s blood
yield significantly, even though old trees produced slight
less than young trees upon the fungal elicitation. For
example, a tree aged more than 100 years with a trunk
diameter of 1.2 m had a yield of 3.7 g/inoculation site,
which was 14% lower than that of a 20-year-old tree (P =
0.09, n = 10). In addition, the inoculation position of the
fungus on the stem (trunk or branch) did not significantly
affect the yield as long as the stem was healthy and
vigorous. Furthermore, the removal of the small pieces of
resinous wood to harvest the induced dragon’s blood did
not significantly affect the growth of the trees. When we
are preparing this manuscript all the tested trees are still
living.
Chemical constituent of the induced dragon’s blood
We developed a UPLC method to compare the chemical
constituents of fungal induced dragon’s blood with the
natural one through establishing chromatographic
fingerprint and simultaneously determining the content of
loureirins a and b. As shown in Figure 2, the UPLC
fingerprints of the fungal-induced dragon’s blood by
strains YM-2617 and YM-6113, and the natural standard
were very similar, indicating the chemical ingredient
similarity among them. The content of loureirin a in the
dragon’s blood induced by YM-2617 and YM-6113 was
significantly higher by 44 and 30% than that of the
standard (P < 0.01), respectively (Figure 3); while the
content of loureirin b of them was similar.
Comparison of the antimicrobial activity between
natural and fungal-induced dragon’s blood
The dragon’s blood had a higher activity against fungi
than against bacteria (Figure 4). At all the test
concentrations, the fungal (YM-6113)-induced dragon’s
blood had a significantly higher activity against its
inducing fungus YM-6113 than the natural dragon’s blood
(P < 0.03, n=4); there was no significant difference
between the fungal-induced dragon’s blood and the
natural dragon’s blood against Pseudomonas aeruginosa.
2636 J. Med. Plant. Res.
0
1
2
3
4
5
0 3 6 9 12
Time post fungus inoculation (week)
Dragon's blood dw
(g/inoculation site)
control 1
control 2
YM-6113
YM-2617
Figure 1. Kinetics of dragon’s blood production in the wounded stem xylem of D. cochinchinensis
trees induced by the inoculation of living F. oxysporum mycelia (strains YM-6113 and YM-2617).
Holes with a surface area of about 0.5 × 0.5 cm2 were punched in the stem xylem. The agar
media with activated mycelia were cut into 0.5 × 0.5 × 0.3 cm3 chips. Each hole was inoculated
with a microbial chip, with the mycelia facing and contacting the bottom of the hole. Afterwards the
inoculation site (wound) was kept moist by sealed with a plastic membrane. The tested trees were
about 20 years old with a trunk diameter of about 5 cm when the experiments were carried out.
Control 1, wounding alone without keeping moist; control 2, wounding and kept the wound moist.
Values were mean ± standard deviation (n = 20).
At 200 and 2000 mg/l, the fungal-induced dragon’s blood
had a significantly higher antimicrobial activity against
YM-2617, Aspergillus niger and Escherichia coli than the
natural dragon’s blood (P < 0.04, n=4). The fungal-
induced and the natural dragon’s blood had no significant
difference against P. aeruginosa. Figure 5 shown the
higher anti- Fusarium oxysporum YM-2617 activity of the
dragon’s blood induced by F. oxysporum YM-6113 than
that of natural standard dragon’s blood.
Identification of strains YM-2617 and YM-6113
These two isolates were identified as two strains of F.
oxysporum (Leslie and Summerell, 2006) by morphology
and nuclear rDNA ITS sequence analysis. The
characteristics of the two strains on CLA were very
similar being in accordance with F. oxysporum. The major
differences between the two strains were as follows. On
PDA, mycelium of strain YM-6113 was deeper purple or
purple with pale magenta pigment occurred in the bottom
of the agar plate; however, mycelium of strain YM-2617
was white with a little of purple pigment occurred the
bottom of the plate. The highest sequence similarity of
the nuclear rDNA ITS of the two strains to that of F.
oxysporum was 99.6% (Figure 6). The two strains have
been deposited in Yunnan Institute of Microbiology,
Kunming, China.
DISCUSSION
The present work has clearly demonstrated that
inoculation with the mycelia of F. oxysporum YM-2617
and YM-6113 significantly improved dragon’s blood
production (Figure 1). At the same time, dragon’s blood
had antimicrobial activity (Figure 4). These results
indicate that dragon’s blood is a special type of
phytoalexins and that the formation of dragon’s blood is a
particular stress response. F. oxysporum is a common
plant pathogenesis with a variety of hosts (Leslie and
Summerell, 2006). Why F. oxysporum isolates other than
the other 18 isolates induce dragon’s blood production is
unclear. Differently, Cytosphaera mangiferae is involved
in the formation of agarwood in Aquilaria malaccensis;
while Melantos flavolives is believed to play a similar role
in Alligator sinensis
(http://www.cropwatch.org/agarwood.htm).
To our knowledge, we for the first time have confirmed
that F. oxysporum induced dragon’s blood formation in D.
cochinchinensis. The fungal induced dragon’s blood had
a similar chemical constituent as that of the natural
Res. Wang et al. 2637
A
B
AU
0. 0 0
0. 0 5
0. 1 0
0. 1 5
0. 2 0
0. 2 5
0. 3 0
0. 3 5
? ?
0. 0 0 1.0 0 2. 0 0 3 .0 0 4 .0 0 5 .0 0 6 .0 0 7 .00 8 . 0 0 9.0 0
1 0. 0 0
A
B
AU
0 .0 0
0 .0 5
0 .1 0
0 .1 5
0 .2 0
0 .2 5
0 .3 0
? ?
0 .0 0 2 .0 0 4 . 0 0 6. 0 0 8. 0 0
1 0. 0 0
A
B
AU
0.00
0.05
0.10
0.15
0.20
0.25
0.30
? ?
0.00 2.00 4.00 6.00 8.00
10. 00
a
b
c
Retention time (min)
Figure 2. Chemical constituent similarity among the natural standard dragon’s blood (a), the dragon’s
blood induced by F. oxysporum YM-6113 (b) and by F. oxysporum YM-2617 (c) analyzed by UPLC at
210 nm. Peak A and B representing loureirin a and b, respectively.
Rentention time (min)
10.00
2638 J. Med. Plant.
0
0.5
1
1.5
2
2.5
3
Standard
control
YM-2617
YM-6113
mg/g
Loureirin a
Loureirin b
Figure 3. Content of loureirins a and b in the dragon’s blood induced by F.
oxysporum YM-2617 and YM-6113, and the natural dragon’s blood. The tested
trees were about 20 years old with a trunk diameter of about 5 cm when the
experiments were carried out. Control, wounding and kept the wound moist but
without microbial inoculation. Values were mean ± standard deviation (n = 4).
0
20
40
60
80
100
120
6113-
I
6113-
N
2617-
I
2617-
N
AN-I AN-N SC-I SC-N BS-I BS-N EC-I EC-N PA-I PA-N
Inhibition (%)
20
200
2000
Figure 4. Comparison of the anti-microbial activity between the dragon’s blood induced by fungal stain F. oxysporum YM-6113 (I)
and natural standard dragon’s blood (N) against different microbial strains (6113, F. oxysporum YM-6113; 2617, F. oxysporum
YM-2617; AN: A. niger; SC: S. cerevisiae; BS: B. subtilis; EC: E. coli; PA: P. aeruginosa). 20, 200 and 2000: the concentration of
dragon’s blood in the medium was 20, 200 and 2000 mg/l (added by a 5% stock solution in 95% ethanol, w/v), respectively. The
negative control, which received the same amount of ethanol as added in the treatment with 2000 mg/l dragon’s blood but without
dragon’s blood, did not have any inhibition effect (data not shown). Values were mean ± standard deviation (n = 4).
dragon’s blood as analyzed by UPLC. In addition, the
induced dragon’s blood had a similar or higher
antimicrobial activity than that of the natural dragon’s
blood. These results indicate that inoculation with the
F. oxysporum isolates could be used to artificially induce
and control dragon’s blood production. The biological
technology has the possibility to induce dragon’s blood at
any time and at any selected part of the trees such as
mg/g
Standard
Control
YM
-
2617
YM
-
6113
Inhibition (%)
Res. Wang et al. 2639
Figure 5. Comparison of the antifungal activity between natural standard dragon’s blood (A) and the induced one by
F. oxysporum YM-6113 (B) against F. oxysporum YM-2617. 20, 200 and 2000 indicating 20, 200 and 2000 mg/l
dragon’s blood in the medium, respectively. The most right plate in the bottom raw was the negative control without
dragon’s blood but received the same amount of ethanol as treatment 2000.
YM-2617 (GU324271)
YM-6113 (GU324272)
Fusarium oxysporum F-T2.1.1-030616-15 (EU364853)
Fusarium nisikadoi NRRL 25179 (U61685)
Fusarium anthophilum NRRL 13602 (U61671)
Fusarium napiforme NRRL 13604 (U34570)
Fusarium subglutinans NRRL 22016 (U34559)
Fusarium begoniae NRRL 25300 (U61673)
Fusarium brevicatenulatum NRRL 25446 (U61675)
Fusarium denticulatum NRRL 25302 (U61680)
Gibberella circinata NRRL 25331 (U61677)
Fusarium ramigenum NRRL 25208 (U61684)
Fusarium lactis NRRL 25200 (U61681)
Fusarium sacchari NRRL 13999 (U34556)
Fusarium proliferatum BU1 (EU151489)
Gibberella fujikuroi NRRL 13566 (U34557)
Fusarium solani VKGFS2 (HM102503)
99
68
88
76
79
74
100
61
50
65
0.02
YM-2617 (GU324271)
YM-6113 (GU324272)
Fusarium oxysporum F-T2.1.1-030616-15 (EU364853)
Fusarium nisikadoi NRRL 25179 (U61685)
Fusarium anthophilum NRRL 13602 (U61671)
Fusarium napiforme NRRL 13604 (U34570)
Fusarium subglutinans NRRL 22016 (U34559)
Fusarium begoniae NRRL 25300 (U61673)
Fusarium brevicatenulatum NRRL 25446 (U61675)
Fusarium denticulatum NRRL 25302 (U61680)
Gibberella circinata NRRL 25331 (U61677)
Fusarium ramigenum NRRL 25208 (U61684)
Fusarium lactis NRRL 25200 (U61681)
Fusarium sacchari NRRL 13999 (U34556)
Fusarium proliferatum BU1 (EU151489)
Gibberella fujikuroi NRRL 13566 (U34557)
Fusarium solani VKGFS2 (HM102503)
99
68
88
76
79
74
100
61
50
65
0.02
Figure 6. A phylogenetic tree showing the relationships of the nuclear rDNA sequences among strain YM-2617, YM-6113 and
relative strains of the genera Fusarium and Gibberella. The tree was constructed using the neighbour-joining method. GenBank
accession numbers were given in parentheses. Numbers represent confidence levels (percentages higher than 50% are shown)
from bootstrap resampling with 1000 replicates. Bar, 0.02 substitutions per nucleotide position.
branches and the ideal parts of the trunk that keep a
stock enough for propagating branches for sustainable
production without destroying the valuable endangered
trees and disturbing the environment.
ACKNOWLEDGEMENT
This work was supported by National Natural Science
Foundation of China (30760300).
2640 J. Med. Plant.
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