全 文 ：Effect of Endophytic Fungi AL12 on the
Metabolites Distribution in Organs of
Atraetylodes lancea
Yingxue GAO, Lei LI, Chuanchao DAI*
Jiangsu Engineering and Technology Research Center for Industrialization of Microbial Resources, Jiangsu Key Laboratory for Mi-
crobes and Functional Genomics, College of Life Science, Nanjing Normal University, Nanjing 210046, China
Supported by the National Natural Science Foundation of China (31070443, 30970523);
the National Science Foundation for Talents Training in Basic Science, China
(J1103507); the Project of the Priority Academic Program Development of Jiangsu
Higher Education Institutions, China.
*Corresponding author. E-mail: daichuanchao@njnu.edu.cn
Received: January 18, 2012 Accepted: February 24, 2012A
Abstract [Objective] To examine the effect of endophytic fungi AL12 (Gilmaniella
sp.) on metabolites distribution in organs of Atractylodes lancea. [Method] Endophytic
fungi AL12 was inoculated on Atraetylodes lancea plantlets in tissue culture, and the
distribution of cellulose, hemicellulose, lignin, soluble sugar in leaves and roots of the
inoculated group were detected. The weight of leaves and roots were compared.
Gas Chromatography was used to analyze the volatile oil components. [Result]
Compared with the control group, the average fresh weight and dry weight of leaves
and roots of A. lancea which had been symbiosed with AL12 increased significantly.
The content of lignin and soluble sugar increased in the leaves of the inoculated
group, and the content of cellulose, hemicellulose, lignin, soluble sugar and volatile
oil also increased in roots. [Conclusion] The results indicate that symbiosed with
AL12 is benefit for the development of A. lancea roots and can promote the transfer
and accumulation of the medicinal components to the roots.
Key words Atractylodes lancea; Endophytic fungi; Metabolite; Organ allocation
Agricultural Science & Technology, 2012, 13(4): 798-803
Copyright訫 2012, Information Institute of HAAS. All rights reserved Plant Protection
A tractylodes lancea has a widerange of medicinal value [1]. A.lancea, a commonly name for
Maoshancangzhu, is a genuine re-
gional drug screened out from the
widely distributed areas based on
long-term clinical practice. It has ex-
cellent quality and efficacy. In recent
years, the relationship between A.
lancea endophytic fungi and medicinal
ingredients accumulation has received
extensive attention from researchers.
Chen et al. explored the biological di-
versity and ecological distribution of A.
lancea endophytic fungi, as well as the
effect of endophytic fungi on the
volatile oil accumulation in A. lancea[2-6].
Chen et al. studied the effects of endo-
phytic fungus (Phomopsis sp.) on de-
composition of A. lancea litters and
activity of degrading enzymes in soil [7].
Wang et al. reported the antimicrobial
activity of volatile oil [8]. However, there
is no report on the effect of endophytic
fungi on the metabolites distribution in
organs of A. lancea.
This study investigated the effects
of endophytic fungi, which were sym-
biosed with A. lancea, on the distribu-
tion of 5 kinds metabolites in organs of
A. lancea with the aim to resolve the
rules of metabolites distribution in or-
gans of A. lancea to provide refer-
ences for the application of endophytic
fungi in actual production and theoreti-
cal basis for the scientific research and
comprehensive development and uti-
lization of A. lancea.
Materials and Methods
Materials
Experimental materials The tested
A. lancea is the A. lancea plantlets
grown for 30 d in tissue culture [2,9]; en-
dophytic fungi AL12 (Gilmaniella sp.)
strains were isolated and preserved by
the Laboratory for Microbes, College
of Life Sciences, Nanjing Normal Uni-
versity[2].
Experimental reagents MS differ-
entiation medium (MS medium+6-BA
2 mg/L +NAA 0.3 mg/L), MS rooting
medium (MS medium+NAA 0.5 mg/L),
PDA medium (potato 200 g/L, glucose
20 g/L, agar 15 g/L), PD medium (PDA
medium without agar), acetic acid-ni-
tric acid mixture (V ∶V = 4∶1), 80% cal-
cium nitrate solution, glucose, DNS
reagent [ 10 ] , sodium hydroxide , phe -
nolphthalein, 1% acetic acid, 10% sul-
furic acid, 0.1 mol/L potassium dichro-
mate solution, 20% KI solution, 0.5%
starch solution, 0.2 mol/L sodium thio-
sulfate solution, sucrose, concentrated
sulfuric acid, 9% phenol, and chro-
matography pure cyclohexane.
Methods
Measurement of dry weight and wet
weight The mycelia were scraped
from the slant of PDA medium which
preserved AL12 strains, and then
transferred to a new PDA slant. After
cultured for 7 d at 28 ℃ , the mycelia
were scraped and transferred to PD
medium, which was then cultured in
the shaker at 120 r/min, 28 ℃ for 7 d
until the mycelia fermented.
The rooted A. lancea plantlet
which had been cultured for 30 d in tis-
sue culture was taken. And 10 ml of
the mycelial fermentation broth was
directly added to the roots of the ex-
perimental A. lancea plantlet, while the
control group was added with the
same amount of PD medium. The
growth of the plantlet was detected af-
ter culturing for 7 d. Then, the tissue
cultured plantlet was pulled out from
DOI:10.16175/j.cnki.1009-4229.2012.04.040
Agricultural Science & Technology
Vol.13, No.4, 2012 Agricultural Science & Technology
2012
the culture flask, and the scissors was
used to separate the roots and leaves
of the plantlet. Water was used to
wash away the medium in the roots
and then sucked by filter paper, and
the wet weights of leaves and roots
were weighed and recorded. Then the
roots and leaves were placed in differ-
ent plates and dried to constant weight
at 50 ℃, and the dry weights for them
were weighed and recorded.
Measurement of cellulose content
The property of cellulose is very sta-
ble. It is the polymer compound of β-D-
glucose linked by 1,4-glycosidic. Dif-
ferent measuring methods and mea-
suring conditions as well as different
dissolution of hemicellulose and lignin
would result in significant differences
in the measuring results of cellulose
content. This study made some im-
provement on the existing methods,
which measured the cellulose content
by using 72% concentrated sulfuric
acid hydrolysis method[10].
First, 0.1 g of dried A. lancea was
taken and placed in a test tube, added
with 5 ml of acetic acid-nitric acid mix-
ture, and heated in boiling water for 25
min with constantly stirring. After
cooled, the solution was centrifuged.
Discarding the supernatant, the pre-
cipitate was rinsed with dH2O for three
times. Then, 10 ml of 10% sulfuric acid
and 10 ml of 0.1 mol/L potassium
dichromate solution were added to the
precipitate. After fully mixed, the solu-
tion was boiled in the boiling water for
10 min, then it was removed to the
flask, and dH2O was used to rinse the
test tube, which was also poured into
the flask. After cooled, the solution
was added with 5 ml of 20% KI solu-
tion and 1 ml of 0.5% starch solution,
titrating with 0.2 mol/L sodium thiosul-
fate, and the one without KI and starch
solution was set as the blank control.
Each sample repeated for six times,
and the average was taken.
Formula for calculating cellu-
lose content: X=K(a-b)/(n×24)
Where, 24 is the equivalent weight of
1 mol cellulose to the sodium thiosul-
fate; K is the concentration of sodium
thiosulfate (mol/L); a is the volume of
sodium thiosulfate consumed in blank
titration (ml); b is the volume of sodium
thiosulfate consumed by the solution
(ml); n is mass of A. lancea (g).
Measurement of hemicellulose
content In plant cell walls, cellulose
and lignin is formed by the close inter-
twined glycan mixture which is known
as hemicellulose, and hemicellulose is
soluble in dilute acid. In this study, the
2 mol/L HCl was used to hydrolyze
hemicellulose, and then the DNS col-
orimetry was used to quantify the hy-
drolyzed reducing sugar to converse
the hemicellulose content[11].
First, 1.5 g of dried A. lancea was
transferred to a 100 ml Erlenmeyer
flask, added with 60 ml of 2 mol/L HCl,
and extracted at 110 ℃ for 60 min.
Then the extract was filtered by funnel,
and rinsed by dH2O for 4 -5 times.
Keeping the residues, the filtrate was
combined and set to constant volume
of 1 L, and placed for overnight. Then,
1 ml of filtrate was put into the test
tube, and neutralized by 1 ml of 0.6
mol/L NaOH, and then added with 1.5
ml of DNS solution. After fully mixed,
the mixture was heat in the boiling wa-
ter bath for 5 min. After cooled, the
mixture was added with 5 ml of dH2O,
and shook until evenly mixed. The
dH2O was used as the reference solu-
tion. The spectrophotometer was used
to measure the absorbance A at
wavelength of 520 nm, and the reduc-
ing sugar content in the filtrate was
calculated according to the standard
curve. Each samplewas repeated three
times, and the average was taken.
Formula for calculating hemicellu-
lose content:Mb=Wb× 0.9/W0 × 100%
Where, Mb is the hemicellulose
content (×10-2 g/g); Wb is the mass of
reducing sugar (μg); W0 is the mass of
A. lancea (g); 0.9 is the coefficient for
conversing hemicellulose form reduc-
ing sugar.
Measurement of lignin content
Lignin is the chemical composition of
plants, which forms the plant skeleton
together with cellulose and hemicellu-
lose. The accumulation and change
rules of lignin are closely related with
the growth, planting conditions and
character verification of Chinese
herbal medicines[12].
After smashed, the A. lancea was
screened by the 30-mesh sieve, and
then 0.05-0.10 g of A. lancea was put
into a centrifuge tube, added with 10
ml of 1% acetic acid, centrifuged after
fully mixed. The precipitate was rinsed
with 5 ml of 1% acetic acid for 1 time,
and then after immersed in 3-4 ml of
ethanol and ether mixture (volume ra-
tio of 1 ∶1) for 3 min, the mixture was
centrifuged and the supernatant was
discarded. The rinse and immersion
were repeated three times. The precip-
itate in the tube was evaporated to dry-
ness in the boiling water bath, and
then added with 3 ml of 72% sulfuric
acid. After evenly mixed by stirring with
a glass rod, the mixture was placed at
room temperature for 16 h to make the
cellulose fully dissolved. And then, 10
ml of dH2O was added into the tube,
after mixed up by stirring with glass
rod, the mixture was then put in the
boiling water bath for 5 min. After
cooled, 5 ml of dH2O and 0.5 ml of
10% barium chloride solution were
added, which was then centrifuged af-
ter fully mixed. After rinsed with dH2O
for 2 times, the precipitate was added
with 10 ml of 10% sulfuric acid and 10
ml of 0.1 mol/L potassium dichromate
solution, and placed in the boiling wa-
ter bath for 15 min, stirring from time to
time. After cooled, all substances in
the test tube were transferred to a
beaker, and the residual part was
washed up by 15 -20 ml of dH2O.
Then, 5 ml of 20% KI solution and 1 ml
of 0.5% starch solution was added to
the beaker, titrating with sodium thio-
sulfate; the blank sample was sepa-
rately titrated by 10 ml of 10% sulfuric
acid and 10 ml of 0.1 mol/L potassium
dichromate solution as the control.
Each sample repeated three times,
and the average was taken.
Formula for calculating lignin
content: X = K (a - b) / (n × 48)
Where, 48 is the equivalent
weight of 1 mol of C11H12O4 to sodium
thiosulfate; K is the concentration of
sodium thiosulfate (mol/L); a is the
volume of sodium thiosulfate con-
sumed by blank titration (ml); b is the
volume of sodium thiosulfate con-
sumed by solution titration (ml); n is
the mass of A. lancea (g).
Measurement of soluble sugar con-
tent Soluble sugar is the main pho-
tosynthetic products of higher plants
and the main form of carbohydrate
metabolism and temporary storage,
playing an important part in the plant
metabolism. According to the method
of Chen [ 13 ] , exactly 1 g of sucrose
which had been dried to constant
weight at 80 ℃ was taken and dis-
solved by adding with a small amount
of dH2O, and then it was transferred to
a 100 ml volumetric flask. After adding
with 0.5 ml of concentrated sulfuric
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2012
Table 1 Effect of endophytic fungi inoculation on the weight of A. lancea plantlet
Organ Wet weight∥g/plant Dry weight∥g/plant Drying rate∥%
Control group Leaf 1.05 0.13 0.12
Root 1.38 0.19 0.14
Experimental group Leaf 1.86 0.21 0.11
Root 1.58 0.26 0.17
acid, the volume of the solution was
set to the scale by dH2O, fully mixed.
Exactly 1 ml of the above solution was
taken and put in the 100 ml volumetric
flask, and the volume was metered by
add dH2O, obtaining 100 mg/L stan-
dard sucrose solution. Then, 0, 0.2,
0.4, 0.6, 0.8, 1.0 ml of the standard
sucrose solution were respectively
taken, by adding dH2O to 2 ml, the
standard colorimetric series containing
0, 20, 40, 60, 80, 100 μg of sucrose
were obtained, which were then added
with 1 ml of 90% phenol solution one
by one. After fully mixed, 5 ml of con-
centrated sulfuric acid was quickly
added dropwise, after fully mixed, the
solution was placed at room tempera-
ture for 30 min. Except containing no
sugar, the control group is the same as
experimental group in other treat-
ments. The absorbance at the wave-
length of 485 nm was measured, and
the standard curve was plotted with
the sucrose content as the abscissa
and absorbance as the vertical axis.
After smashed, 0.8 g of A. lancea
was weighed and placed in the gradu-
ated tube, added with 20 ml of dH2O,
and then sealed with plastic film and
extracted in boiling water for 2 times,
each time for 30 min. The extract was
filtered into the 50 ml volumetric flask,
and the tube and residues were re-
peatedly rinsed and the constant vol-
ume was fixed to the mark. Then, 0.5
ml of sample solution was put into the
test tube, diluted to 2 ml by adding
dH2O, and then the absorbance of the
sample was measured according to
the above method. The soluble sugar
content in the A. lancea was calculated
according to the standard curve.
Measurement of volatile oil content
After smashed, exact 1 g of A.
lancea was taken and immersed in 4
times volume of cyclohexane for 8 h,
and then it was extracted by ultrasoni-
cation at 30 ℃ for 15 min. After cen-
trifuged at 5 000 r/min for 5 min, the
supernatant was taken, and added
with anhydrous sulfatesodium. Then
the gas chromatography was used to
analyze the volatile oil content[14].
Results and Analysis
Effect of endophytic fungi inocula-
tion on the weight of A. lancea
plantlet
As shown in Table 1, compared
with the control group, the average wet
weight and dry weight of leaves in the
experimental group increased 77.14%
and 61.54%, respectively; the wet wei-
ght and dry weight of roots in experi-
mental group increased 14.49% and
21.43% , respectively. And compared
with the control group, the drying rate
of leaves in the experimental group
decreased 1% , while the root in-
creased 3% . Within the same growth
time, after adding with AL12, the total
weight increased more in the leaves
than in the roots of the A. lancea
plantlet.
Effect of endophytic fungi inocu-
lation on the cellulose content in
A. lancea
The proportion of cellulose in per
gram of the sample was as shown in
Fig.1. Compared with the control, the
cellulose content in the leaves of the
experimental group was 6.08% less,
while in the roots it was 3.38% more.
In the control group, the cellulose con-
tent in the leaves was 2.48% higher
than in the roots, while in the inoculat-
ed group, the cellulose content in the
roots was 6.98 % higher than in the
leaves.
Effect of endophytic fungi inocula-
tion on the hemicellulose content in
A. lancea
Within the range of 0 -1 000 μg,
the standard curve was plotted with
the glucose content as the horizontal
axis and the absorbance at the 540
nm (A540) as the vertical axis, obtain-
ing the regression curve equation: Y =
0.000 9X-0.012 4, R2 = 0.999 4.
According to the glucose standard
curve, the hemicellulose contents in
the samples were as shown in Fig.2.
Compared with the control group, the
hemicellulose content in the leaves of
the experimental group decreased
0.34%, while the hemicellulose content
in the root increased 3.17%. The hem-
icellulose content in the leaves of the
control group was 0.26% higher than
in the roots, while in the experimental
group, the hemicellulose content was
3.25% higher in the roots than in the
leaves.
Effect of endophytic fungi inocu-
lation on the lignin content in
A . lancea
Lignin contents in the testing
samples were as shown in Fig.3. After
Fig.2 Effect of inoculation treatment on the content of hemicellulose in A. lancea
Fig.1 Effect of inoculation treatment on the content of cellulose in A. lancea
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Vol.13, No.4, 2012 Agricultural Science & Technology
2012
Control leaf and control root represent the control group; test leaf and test root represent the
experimental group.
Fig.5 Effect of inoculation treatment on the content of four major active compounds in
volatile oil
Control leaf and control root represent the control group; test leaf and test root represent the
experimental group.
Fig.4 Effect of inoculation treatment on the content of soluble sugar in A. lancea
Control leaf and control root represent the control group; test leaf and test root represent the
experimental group.
Fig.3 Effect of inoculation treatment on the content of lignin in A. lancea
inoculation, compared with the control,
the lignin contents in the leaves and
roots of the experimental group were
increased by 2.89% and 5.49% , re-
spectively. The lignin content in the
roots of the control group was 6.93 %
higher than in the roots, while in the
experimental group, the lignin content
in the roots was 4.33% higher than in
the leaves. Therefore, AL12 can pro-
mote the increase of lignin content in
the roots and leaves of A. lancea, and
the effect is more significant in the
leaves than in the roots.
Effect of endophytic fungi inocula-
tion on the soluble sugar content in
A. lancea
Within the range of 0-100 μg, the
standard curve was plotted with the
sucrose content as the horizontal axis
and the absorbance at the 485 nm
(A485) as the vertical axis, obtaining the
regressioncurveequation:Y=0.0049X+
0.010 6, R2 = 0.999 1.
According to the measured ab-
sorbance value and the standard
curve of sucrose, the soluble sugar
content in each sample was shown in
Fig.4. Compared with the control, the
soluble sugar content in the leaves
and roots of the experimental group
were all increased, 3.22% and 0.30%,
respectively. The difference between
the soluble content in the roots and
leaves of the control group was
11.16%, while it was 8.24% in the ex-
perimental group. The results show
that inoculation treatment can increase
the soluble sugar content in the leaves
of A. lancea, but its effect on the root is
not obvious.
Changes of volatile oil content in
A. lancea
Atractylone, atractylol, β-eu-
desmol and atractylodin are the 4 main
active compounds in the volatile oil of
A. lancea, so the changes of these
four substances were detected (Fig.5).
Compared with the control group, the
total contents of these four substances
in the leaves of the experimental group
were decreased, while increased in
the roots. As for the content of single
substance, the atractylone and atra-
ctylol content in the leaves of the ex-
perimental group was smaller than in
the control group, while higher than the
control in the roots; β-eudesmol con-
tent was exactly contrary to atracty-
lone and atractylol; the change of the
atractylodin content was the biggest a-
mong the 4 compounds that it content
in the leaves and roots of the experi-
mental group were all higher than the
control. Since the root is the medicinal
part of the A. lancea, therefore, inocu-
lation of endophytic fungi AL12 in-
creased the contents of atractylone, a-
tractylol and atractylodin while de-
creased the β-eudesmol content,
which was consistent with the volatile
oil characteristics of genuine regional
A. lancea.
Conclusion and Discussion
The results indicate that sym-
biosed with AL12 is benefit for the de-
velopment of A. lancea roots and can
promote the transfer and accumulation
of the medicinal components to the
roots.
Biomass changes in A. lancea
Endophytic fungi can promote the
absorption of K, Mg, Cu, Zn, F, B, and
Ca by the plants, some of which can
only increase dry weight and biomass
of the plant [15]. Wu et al. isolated a
dark-septate endophyte fungus EF-M
from Saussurea involucrate, found that
by inoculating the hosts, there were no
profound effects of endophyte EF-M
on plant root development, but signifi-
cant differences were detected in plant
height and shoot dry weight between
the treatments[16].Malinow-skiet al. found
that in the P efficiency soil, compared
with the uninfected plants, the absorp-
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Agricultural Science & Technology
Agricultural Science & Technology Vol.13, No.4, 2012
2012
tion of N and Mg in the endophyte in-
fected plants increased the same as P,
and also found that nutrients uptake
rates in roots were affected by endo-
phyte infection in leaves[17]. Some ele-
ments play the bioremediation role in
plant growth, for example, Wise et al.
promoted the absorption of Zn and Cd
by mycorrhizal fungi and bacteria sym-
biosis[18].
Yuan et al. studied the physiologi-
cal characteristics of endophytic fun-
gus B3, found that it can secrete hor-
mones IAA and ABA, and its fermen-
tation broth contains VB1 and the
mycelium contains 16 kinds of amino
acids and a variety of fatty acids, sug-
gesting that there may be material and
energy exchanges between the endo-
phytic fungi B3 and the host rice [19].
There are many reasons for endo-
phytes B3 to promote the growth of
rice, among which secretion of plant
hormones is a more direct reason; the
synthesis of mino acids, vitamins and
fatty acids, which are essential for rice,
can also promote the growth.
This test shows that the combin-
ing fastness between tissue culture
plantlet and the medium of the experi-
mental group is greater than the con-
trol group, and AL12 can promote the
growth of roots and leaves in the ex-
perimental group. The reason might be
that AL12 can promote the plantlet ab-
sorb inorganic elements by promoting
the combination between the roots of
A. lancea plantlet and media combina-
tion; it might also be that AL12 can se-
crete growth-promoting substances
such as growth hormone, which pro-
mote the growth of the A. lancea. The
results show that the AL12 can in-
crease drying rate in the roots of the A.
lancea plantlet, indicating that sym-
biosis with the fungi can promote the
accumulation of dry matter in the plant
roots, and the reason may be that fun-
gi can stimulate secondary metabolic
activities in plant. Increasing the drying
rate of aseptic plantlet is of important
practical significance. AL12 significant-
ly increases the drying rate of tissue
culture plantlet without affecting the
normal growth, which is equivalent to
improve the production of tissue cul-
ture plantlet.
Carbohydrate content changes in
A. lancea
Claydon et al. isolated indole
acetic acid (IAA) and other indole
derivatives from the Baansa epichloe
which was grown in the medium con-
taining tryptophan [20 ] , and they sug -
gested that endophytic fungi can
change the hormone metabolism in
the plants by producing hormones like
auxin, promoting plant growth. Zhang
et al. isolated 5 species of endophytic
fungi from Noectochilus roxburghii and
some other medicinal plants, found
that these 5 species of endophytic
fungi can produce one or several kinds
of plant hormones, and gibberellic acid
(GA), indole acetic acid (IAA), abscisic
acid (ABA), zeatin (Z) and zeatin ribo-
side (ZR) were detected from the
mycelia and its culture solution [21].
Therefore, it is thought that endo-
phytes can promote the plant growth
by changing the hormones metabolism
of plant. The tested AL12 may also
have similar functions that promote the
root growth of A. lancea by plant hor-
mones. The reason for the mechanism
may be that after infecting the A. lan-
cea plantlet, the AL12, as endophytic
fungi, can simulate the cell division
and cell wall thickening in the root sys-
tem of the plants; or that AL12 can
produce certain metabolites to pro-
mote the growth of A. lancea root while
inhibit the leaf growth, and the sensi-
tivity of metabolites to stems and
leaves is greater than the roots; or that
AL12 change the source and library
distribution relationship of glucose in
the A. lancea plantlet of tissue culture,
strengthening the root competition. As
a principle composition in plant cell
walls, the increase of cellulose content
can be used as an indicator to deter-
mine the growth.
Hemicellulose is a heterogeneous
polymer composed by several different
types of monosaccharides, which are
pentose and hexose, including xylose,
arabinose, mannose and galactose.
The change of hemicellulose content
is similar to that of cellulose. As one of
the components of cell wall, the raw
synthetic material is carbonhydrate.
The reasons for the effect of endophyt-
ic fungi AL12 on the hemicellulose con-
tent in A. lancea may also similar to
that for cellulose content.
The green leaves or green stems
are the part can produce soluble sugar
in plants, while the non-green tissues
such as roots are the parts to store or
consume sugar. In this study, the sol-
uble content in the leaves of experi-
mental A. lancea increases while there
is no significant changes in the roots,
which might because that AL12 pro-
motes the synthesis of sugars in the
plantlet leaves of A. lancea, but does
not enhance the transportation, or that
as a new soluble sugar consumption
site, AL12 shares the soluble sugar in
the roots from the leaves.
As two components of plant cell
walls, the cellulose and hemicellulose
contents in the cell walls increase after
the invasion of AL12 to roots which en-
hance the cells proliferation in roots.
Although the soluble sugar synthesis
increases in leaves, but the cell divi-
sion is not strong or even inhibited, re-
sulting in lower cell wall synthesis than
the control. Lignin is a phenolic sec-
ondary metabolite, which has different
synthesis pathway with cellulose and
hemicellulose, thus there is no neces-
sary relationship between its content
changes and cellulose and hemicellu-
lose.
Lignin content changes in A. lancea
Lignin is a complex phenolic poly-
mer composed with 4 kinds of alcohol
monomer (P-coumaric alcohol, conif-
eryl alcohol, 5-hydroxy-coniferyl alco-
hol, sinapic alcohol) which can link the
cells. It is one of the constituents that
make up plant cell walls. The reason
for the changes of lignin content in the
endophytic fungi AL12 inoculated
A . lancea may be that AL12 produce
some substances which can affect the
distribution of raw materials for lignin
synthesis in plants, synthesizing more
flow leaves when increasing the lignin
synthesis of the whole plant.
Volatile oil content changes in
A. lancea
The volatile oil is generated in
leaves of A. lancea, then transported
to the roots and stored there. After the
inoculation treatment, except β-eu-
desmol which is decrease slightly, the
other 3 kinds of active compounds of
volatile oil in the root of the experi-
mental group all present upward
trends. The speculated reason is that
symbiosis with the AL12 promotes the
syntheses of atractylone, atractylol
and atractylodin, and promotes the
transportation of volatile oil to the
roots.
References
[1] LIU XH(刘晓辉), LI UXJ(刘显军), CHEN
J (陈静). Natural properties and biologi-
802
Agricultural Science & Technology
Vol.13, No.4, 2012 Agricultural Science & Technology
2012
内生真菌 AL12对茅苍术代谢产物器官分配的影响
高映雪，李 蕾，戴传超 * （南京师范大学生命科学学院，江苏省微生物资源产业化工程技术研究中心，江苏省微生物与功能基因组学重点实验
室，江苏南京 210046）
摘 要 [目的]研究内生真菌孔球孢霉 AL12号菌株对茅苍术组培苗生长及其代谢产物器官分配的影响。[方法]将内生真菌 AL12与茅苍术组培
苗进行共培养，比较感染前后茅苍术叶、根重量差异，检测接种 AL12的组培苗中纤维素、半纤维素、木质素以及可溶性糖在叶片和根中的分布，并
利用气相色谱分析挥发油组分。[结果]茅苍术与 AL12共生后，与对照组相比，其组培苗叶片和根的鲜重和干重均有明显增加，且叶片木质素和可
溶性糖含量增加，根部纤维素、半纤维素、木质素、可溶性糖及挥发油含量均有提高。[结论]该研究结果表明与 AL12共生有利于茅苍术根部发育
且能促使药用成分向根中转移、积累。
关键词 茅苍术；内生真菌；代谢产物；器官分配
基金项目 国家自然科学基金（31070443，30970523）；国家基础科学人才培养基金（J1103507）；江苏高校优势学科建设工程资助项目。
作者简介 高映雪（1988-），女，江苏淮安人，研究方向：微生物生态学，E-mail:littlemousebetty@163.com。*通讯作者，教授，博士，博士生导师，从事
微生物生态学研究，E-mail: daichuanchao@njnu.edu.cn。
收稿日期 2012-01-18 修回日期 2012-02-24
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Responsible editor: Na LI Responsible proofreader: Xiaoyan WU
cal functions of Rhizoma atractylodis(苍
术的性质及生物学功能)[J]. Hubei Agri-
cultural Sciences(湖北农业科学), 2010,
49(6): 1476-1478.
[2] CHEN JX(陈佳昕), DAI CC(戴传超), LI
X(李霞), et al. Endophytic fungi screen-
ing from Atracty lancea and inoculating
into the host plantlet(茅苍术内生真菌的
分离鉴定及在组培苗中的回接)[J]. Gui-
haia(广西植物), 2008, 28(2): 256-260.
[3] CAO YM(曹益鸣 ), TAO JH(陶金华 ),
JIANG S(江曙 ), et al. Biodiversity and
ecological distribution of endogenous
fungi in Atractylodes lancea(茅苍术内生
真菌生物多样性与生态分布研究 ) [J].
Journal of Nanjing University of Tradi-
tional Chinese Medicine (南京中医药大
学学报), 2010, 26(2): 137-139.
[4] FANGF(方芳), DAICC(戴传超), ZHANG
B(张波), et al. Establishment of suspen-
sion cell line of Atractylodes lancea and
effect of endophytic fungal elicitors on
its essential oil accumulation (茅苍术悬
浮细胞系建立及内生真菌诱导子对其挥
发油积累的影响)[J]. Chinese Tradition-
al and Herbal Drugs (中草药), 2009, 40
(3): 452-455.
[5] ZHANGB(张波), DAI CC(戴传超), FANG
F(方芳 ), et al. Effects of three species
endophytic fungi on Atractylodes lancea
growth and its essential oil composition
(三种内生真菌对茅苍术组培苗的生长
及主要挥发油成分的影响)[J]. Chinese
Journal of Ecology (生态学杂志), 2009,
28(4): 704-709.
[6] TAO JH(陶金华), PU XL(濮雪莲), JIANG
S(江曙). Effect of endophytic fungal elic-
itors on growth and atractylodin accu-
mulation of cell suspension cultures of
Atractylodes lancea(内生真菌诱导子对
茅苍术细胞生长及苍术素积累的影响)
[J]. China Journal of Chinese Materia
Medica (中国中药杂志 ), 2011, 36 (1):
27-31.
[7] CHEN Y(陈晏), DAI CC(戴传超), WANG
XX(王兴祥), et al. Effects of endophytic
fungus (Phomopsis sp.) on decomposi-
tion of plant(atractylodes lancea (thunb)
dc) litters and activity of degrading en-
zymes in soil(施加内生真菌拟茎点霉对
茅苍术凋落物降解及土壤降解酶活性的
影响)[J]. Acta Pedologica Sinica(土壤学
报), 2010, 47(3): 537-544.
[8] WANG Y(王宇), DAI CC(戴传超), CHEN
Y(陈晏). Antimicrobial activity of volatile
oil from Atractylodes lancea against
three species of endophytic fungi and
seven species of exogenous fungi(茅苍
术挥发油对三种内生真菌及七种外源真
菌的抑菌活性的研究)[J]. Chinese Jour-
nal of Applied Ecology(应用生态学报 ),
2009, 20(11): 2778- 2784.
[9] LI XT(李西腾), WU YY(吴沿友). Tissue
culture of Atractylodes lancea (Thunb.)
DC.(茅苍术的组织培养和快速繁殖)[J].
Research and Practice of Chinese Mde-
icines(广西热带农业), 2006(2): 33-34.
[10] XIONG SM(熊素敏), ZUO XF(左秀凤),
ZHU YY(朱永义). Determination of cel-
lulose, hemi-cellulose and ligin in rice
hull(稻壳中纤维素、半纤维素和木质素
的测定)[J]. Cereal & Feed Industry (粮
食与饲料工业), 2005(8): 40-41.
[11] ZHAO XF(赵小锋), ZHANG J(张军), LI
SB(李素波), et al. Study on quantitative
analysis method of flax fiber chemical
composition(亚麻纤维化学成分定量分
析方法研究)[J]. Shanghai Textile Sci-
ence & Technology (上海纺织科技 ),
2008, 36(2): 49-51.
[12] LI J(李靖), LI S(李珊), GU M(顾敏), et
al. Determination of lignin content in
tiny panax ginseng by UV spectropho-
tometry(紫外分光光度法测定微量人参
木质素的含量 )[J]. Journal of Chinese
Medicinal Materials(中药材), 2006, 29
(3): 239-241.
[13] CHEN KK (陈克克 ). Determination of
soluble sugar and reducing sugar in
the root of lycopus lucidus turcz(地瓜
儿可溶性糖和还原糖的含量测定 ) [J].
Journal of Xi’an University of Arts and
Science: Natural Science Edition(西安
文理学院学报), 2009, 12(1): 39-42.
[14] ZHANGB(张波),DAICC(戴传超), FANG
F(方芳), et al. Effects of three species
endophytic fungi on Atractylodes
lancea growth and its essential oil
composition(三种内生真菌对茅苍术组
培苗生长及主要挥发油成分的影响)[J].
China Journal of Chinese Materia
Medica (中国中药杂志), 2009, 28(4):
704-709.
[15] MONZOM A, AZOM R. Relevance of
mycorrhizal fungal origin and host
plant genotype to inducing growth and
nutrient uptake in Medicago species
[J]. Agr Ecosyst Enciron, 1996, 60(1):
9-15.
[16] WU LQ, GUO SX. Interaction between
an isolate of dark-septate fungi and its
host plant Saussurea involucrate [J].
Mycorrhiza, 2008(18): 79-85.
[17] MALINOWSKID P, ALLOUSH GA, BE-
LESKY DP. Leaf endophyte Neoty-
phodium coenophialum modifies min-
eral uptake in tall fescue[J]. Plant Nutr,
1999(22): 1335-1349.
[18] WISE DL, TRANTOLO DJ, CICHON
EJ, et al. Bioremediation of contami-
nated soils [M]. New York: Marcel
Dekker, 2000.
[19] YUANZL(袁志林), DAI CC(戴传超), SHI
Y(史央), et al. Mechanism of an endo-
phytic fungi’s stimulation on growth of
rice(内生真菌 B3 促进水稻生长的机理
研究)[J]. Jiangsu Agricultural Sciences
(江苏农业科学), 2004(2): 10-13.
[20] CLAYDON N, GROVEJ F, POPLEM.
Elm bark beetle boring and feeding de-
terrents from Phomopsis oblonga [J].
Phytochemistry, 1985, 24: 937-943.
[21] ZHANG JH(张集慧 ), WANG CL(王春
兰 ), GUO SX(郭顺星 ), et al. Studies
on the plant hormones produced by 5
species of Endophytic fungi isolated
from medicinal plants (Orchidacea)(兰
科药用植物的 5 种内生真菌产生的植
物激素)[J]. Acta Academiae Medicinae
Sinicae(中国医学科学院学报 ), 1999,
21(6): 460-465.
803