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Mycorrhizal Fungi Promote Growth and Nitrogen Utilization by Dendrobium nobile (Orchidaceae)

菌根真菌促进金钗石斛的生长及氮利用



全 文 :菌根真菌促进金钗石斛的生长及氮利用
王秋霞1ꎬ2ꎬ 严  宁1ꎬ 纪大干1ꎬ 李树云1ꎬ 胡江苗3ꎬ 胡  虹1∗
( 1 中国科学院昆明植物研究所资源植物与生物技术重点实验室ꎬ 云南 昆明  650201ꎻ 2 中国科学院
大学ꎬ 北京  100049ꎻ 3 中国科学院昆明植物所植物化学与西部植物资源持续利用
国家重点实验室ꎬ 云南 昆明  650201)
摘要: 菌根在兰科的生命周期和进化史上起着关键作用ꎮ 兰科中大多数是附生兰ꎬ 但它们的菌根研究相对
缺乏ꎮ 为了探讨菌根对附生兰的影响ꎬ 本研究用金钗石斛 (Dendrobium nobile) 与通过形态学特征和分子
生物学鉴定的分属于瘤菌根菌属 (Epulorhiza) 的 S1 和胶膜菌属 (Tulasnella) 的 S3 真菌共培养ꎮ 共培养
结果表明ꎬ S1和 S3与金钗石斛形成了共生关系ꎬ 且不同程度地促进了其生长ꎮ 15N 稳定同位素标记实验
证实ꎬ S1菌株显著促进了金钗石斛对有机氮的利用ꎬ 而 S3 菌株没有显著的促进作用ꎮ 同时ꎬ S1 和 S3 真
菌均能提高金钗石斛中石斛碱的含量ꎮ 研究结果表明ꎬ 菌根真菌能促进附生兰幼苗的生长、 有机氮的利用
和次生代谢产物的积累ꎮ
关键词: 金钗石斛ꎻ 菌根真菌ꎻ 生长ꎻ 15Nꎻ 石斛碱
中图分类号: Q 945ꎬ Q 948􀆰 12        文献标识码: A        文章编号: 2095-0845(2014)03-321-10
Mycorrhizal Fungi Promote Growth and Nitrogen Utilization
by Dendrobium nobile (Orchidaceae)
WANG Qiu ̄Xia1ꎬ2ꎬ YAN Ning1ꎬ JI Da ̄Gan1ꎬ LI Shu ̄Yun1ꎬ HU Jiang ̄Miao3ꎬ HU Hong1∗
(1 Key Laboratory of Economic Plants and Biotechnologyꎬ Kunming Institute of Botanyꎬ Chinese Academy of Sciencesꎬ
Kunming 650201ꎬ Chinaꎻ 2 University of Chinese Academy of Sciencesꎬ Beijing 100049ꎬ Chinaꎻ 3 State Key
Laboratory of Phytochemistry and Plant Resources in West Chinaꎬ Kunming Institute of Botanyꎬ
Chinese Academy of Sciencesꎬ Kunming 650201ꎬ China)
Abstract: Mycorrhizal associations play a key role in the life cycle and evolutionary history of orchids. Although
most orchid species are tropical and epiphyticꎬ their mycorrhizae are poorly understood compared with those of tem ̄
perateꎬ terrestrial orchids. To investigate the influences of such fungi on photosyntheticꎬ epiphytic orchidsꎬ we inoc ̄
ulated seedlings of Dendrobium nobile with Epulorhiza sp. (S1) or Tulasnella sp. (S3). These fungi had been identi ̄
fied based on their morphological and molecular characters. Both S1 and S3 formed symbiotic associations with our
seedlingsꎬ promoting their growth and development to various degrees. Results from signature experiments with the
15N stable isotope suggested that the utilization of organic nitrogen by orchid seedlings was significantly improved by
S1ꎬ but not by S3. Dendrobine contents were significantly higher in all inoculated seedlings. Our findings demon ̄
strate that these mycorrhizal fungi enhance plant growthꎬ their utilization of organic nitrogenꎬ and the accumulation of
secondary metabolites in this epiphytic orchid species.
Key words: Dendrobium nobileꎻ Mycorrhizal fungiꎻ Growthꎻ 15Nꎻ Dendrobine
  Orchidaceaeꎬ the largest plant family in the worldꎬ is estimated to comprise more than 25 000 species.
植 物 分 类 与 资 源 学 报  2014ꎬ 36 (3): 321~330
Plant Diversity and Resources                                    DOI: 10.7677 / ynzwyj201413117
∗ Author for correspondenceꎻ E ̄mail: huhong@mail􀆰 kib􀆰 ac􀆰 cn
Received date: 2013-05-23ꎬ Accepted date: 2013-08-20
作者简介: 王秋霞 (1979-) 女ꎬ 在读博士研究生ꎬ 主要从事兰科植物菌根研究ꎮ E ̄mail: wangqiuxia@mail􀆰 kib􀆰 ac􀆰 cn
Because orchid seeds are minute and contain few
stored food reservesꎬ colonization by a compatible
fungus is essential for germination and / or early
growth and development ( protocorm stage) in the
substrate (Dearnaleyꎬ 2007). Bayman et al. (2002)
have suggested that the development of mycorrhizal
associations was a crucial event in the evolution of
this family. Even though most orchid species are
tropical and epiphyticꎬ their mycorrhizae are poorly
understood compared with those of temperateꎬ terres ̄
trial orchids.
Mycorrhizal infections resulted in significantly
higher N contents in individuals of the green ̄leaved
terrestrial orchid Goodyera repens beyond the proto ̄
corm stage (Cameron et al.ꎬ 2006). Howeverꎬ the
influence that those fungi have on N metabolism in
epiphytic orchids remains unclear. Such orchids live
on trees or rocks with mossesꎬ liverwortsꎬ and ferns
(Suárez et al.ꎬ 2006)ꎬ sites where organic N is a ̄
bundant. This nutrient is a major constituent of sec ̄
ondary metabolitesꎬ proteinsꎬ and nucleic acids. Or ̄
ganic and inorganic N can affect various plant
processesꎬ from growth and development to metabo ̄
lism (Scheible et al.ꎬ 2004). Because alkaloids are
synthesized mostly from amino acids (Haslamꎬ 1986)ꎬ
the higher the level of available Nꎬ the greater the
production of alkaloids.
When devising new strategies of the conserva ̄
tion and artificial propagation of orchidsꎬ researchers
must evaluate the possibly strong impact of these
fungal symbionts ( Liu et al.ꎬ 2010). One of the
most popular orchids within the Dendrobium genusꎬ
D􀆰 nobile Lindl. is an epiphytic and tropical plant
(Mohanty et al.ꎬ 2012). This species is well known
in the pharmaceutical industryꎬ primarily for the for ̄
mation of alkaloid compounds such as dendrobine
(Zha et al.ꎬ 2007). Thereforeꎬ improving our un ̄
derstanding of the mycorrhizal relationships in these
plants would be beneficial for the commercial pro ̄
duction of dendrobine. Our study objectives were to
1) detect whether endophytic fungi from D􀆰 officinale
can form successful mycorrhizal associations with the
roots of D􀆰 nobile seedlings and improve growth of the
host plantsꎬ and 2) investigate whether organic N u ̄
tilization and dendrobine contents in D􀆰 nobile seed ̄
lings can be increased via fungal symbiosis.
1  Materials and methods
1􀆰 1  Isolation of endophytic fungi
Wild plants of Dendrobium officinale were col ̄
lected in April 2009 from a subtropical forest at
Xishuangbanna (21􀆰 7° Nꎬ 100􀆰 8° E)ꎬ Yunnan Prov ̄
inceꎬ China. Healthy roots were selectedꎬ rinsed
with tap waterꎬ and washed again in sterile distilled
water. Once segmentedꎬ they were surface ̄sterilized
by consecutive immersions for 8 to 10 min in 0􀆰 1%
HgCl2ꎬ and then rinsed five times with sterile dis ̄
tilled water. After surface ̄dryingꎬ the root segments
were aseptically cut into approximately 0􀆰 5 to 1 cm
sectionsꎬ and transferred to 9 cm Petri dishes contai ̄
ning potato dextrose agar ( PDA: 20% potatoꎬ 2%
glucoseꎬ and 1􀆰 5% agar). The dishes were incuba ̄
ted in the dark at 25 ℃ until fungal hyphae emerged
from inside the roots. Pure cultures were obtained by
transferring the hyphae onto fresh PDA and storing
them in PDA slant tubes at 4 ℃ . From all of the iso ̄
lates that were naturally presentꎬ we determined that
two in particular—S1 and S3—stimulated growth of
tissue ̄cultured seedlings of D􀆰 nobile through artifi ̄
cial inoculation. Thereforeꎬ they were selected for
further study.
1􀆰 2  Identification of fungi
The S1 and S3 isolates were cultured on a PDA
medium. Micro ̄morphology features of their colonies
were observed with a light microscope (OLYMPUS
CX31)ꎬ and images were made with a video camera
(JVC TK ̄C721EG). Because both S1 and S3 are
sterile in vitroꎬ we used the internal transcribed re ̄
gion (ITS) of the 5􀆰 8S rDNA and the large subunit
gene of mitochondrial rDNA (mtLSU)ꎬ respective ̄
lyꎬ to identify them. Brieflyꎬ DNA was extracted
from 30 ̄day ̄old PDA ̄cultured colonies according to
the cetyltrimethyl ammonium bromide ( CTAB )
method (Doyle and Doyleꎬ 1987). Universal fungal
223                                  植 物 分 类 与 资 源 学 报                            第 36卷
primer combinations ITS1 / ITS4 (Ma et al.ꎬ 2003)
and ML5 / ML6 (Bruns et al.ꎬ 1998) were selected
for ITS and mtLSU amplification. PCR reactions (25
μL) were performed using 2􀆰 5 μL of 10 × buffer
(with Mg2+ )ꎬ 2 μL of 2􀆰 5 mmol􀅰L-1 dNTPꎬ 0􀆰 5
μL of TaqE (2􀆰 5 U)ꎬ 2 μL of 5 μmol􀅰L-1 of each
primerꎬ 2 μL of undiluted DNA template and 14 μL
of ddH2 O. The cycle parameters included denatur ̄
ation at 95 ℃ for 3 minꎻ then 35 cycles of denatur ̄
ation at 94 ℃ for 1 minꎬ annealing at 53 ℃ for 50 sꎬ
and elongation at 72 ℃ for 1 minꎻ followed by a final
extension at 72 ℃ for 7 min. The PCR products were
purified and directly sequenced in an ABI Prism
3730 Sequencer (Applied Biosystemsꎬ Foster Cityꎬ
CAꎬ USA) at the Shanghai Sangon Biological Engi ̄
neering Technology & Services Co.ꎬ Ltd. Their se ̄
quences were aligned by ContigExpress and adjusted
manually. The BLAST search program ( http: / /
www􀆰ncbi􀆰nlm􀆰nih􀆰 gov / BLAST / ) was used for comp ̄
aring their sequence homology with other fungi. If
the sequence to be identified and the vouchered se ̄
quence had >95% similarityꎬ they were assigned to
the same genus (Altschul et al.ꎬ 1990).
1􀆰 3  Inocula of fungi
The S1 and S3 fungi were transferred to 9 cm
Petri dishes containing PDA mediaꎬ where they were
incubated in the dark at 25 ℃ for three weeks.
1􀆰 4  Inoculation experiment
Ripe capsules of Dendrobium nobile were col ̄
lected in September 2010 from a cultivation base in
Puer ( 22􀆰 78° Nꎬ 100􀆰 97° E)ꎬ Yunnan Provinceꎬ
China. Intact capsules were washed with tap water
and surface ̄sterilized with 75% ethanol. They were
then soaked in a 0􀆰 1% HgCl2 solution for 10 minꎬ
and rinsed three times with sterile distilled water.
Afterwardꎬ they were blotted with sterile filter paper
and split. The seeds were sown into culture bottles
(8 cm diam) containing 100 mL of a Harvais medi ̄
um ( autoclaved beforehand at 121 ℃ for 30 min)
and incubated in the greenhouse (12 ̄h photoperiodꎬ
50 μmol􀅰m-2􀅰s-1ꎬ 26±1℃). Germinated seedlings
were aseptically transplanted into new medium and
continually differentiated new seedlings.
Eight uniform seedlings of D􀆰 nobile were im ̄
planted into each new culture bottle (8 cm diam)ꎬ
which contained 100 mL of 1 / 2 MS media supple ̄
mented with 0􀆰 75% sucrose and 0􀆰 75% agar after
weighing (Hou and Guoꎬ 2009). For the inoculation
treatmentꎬ one mycelial plug ( 6 mm diam)ꎬ cut
from the margin of a fungal colony ( either S1 or
S3)ꎬ was placed in the middle of each bottle. As our
control treatmentꎬ the same agar plugs were usedꎬ
but without the fungal addition. Each treatment com ̄
prised 25 replicatesꎬ which were kept in the green ̄
house for two months.
1􀆰 5  Visualization of fungal infection in roots
The fungal hyphae were stained with a chitin ̄
specific dyeꎬ i􀆰 e.ꎬ the wheat germ agglutinin ̄alexa
fluor (WGA ̄AF) 488 conjugate (Molecular Probesꎬ
Karlsruheꎬ Germany). Fungal infections were ob ̄
served as a previously described ( Doehlemann et
al.ꎬ 2008) with some modifications to the technique.
Brieflyꎬ at two months post ̄inoculationꎬ the host
roots were incubated in the staining solution for 60
min. During this periodꎬ the solution was vacuum ̄
infiltrated three times (3 min each) at 25 mm of Hg.
After the samples were rinsed three times in 1×PBS
( phosphate buffer salineꎻ pH 7􀆰 4 )ꎬ they were
transferred into a Propidium Iodide ( PI) solution
(20 μg􀅰mL-1) for 3 minꎬ then rinsed three times in
1×PBS (pH 7􀆰 4). Finallyꎬ the samples were mount ̄
ed on glass slides.
Images were recorded from a laser scanning
confocal microscope (LSCMꎻ OLYMPUS FV1000).
The WGA ̄AF 488 was excited with a 488 ̄nm laser
and detected at 500 to 540 nm. The PI was excited
with a 559 nm laser and detected at 580 to 619 nm.
1􀆰 6  Experimental design for 15N stable isotope
signature in a microcosm
Our experimental microcosms were lidded cul ̄
ture bottles. Each microcosm contained 100 mL of a
1 / 2 MS medium supplemented with 5 mg of 15N ̄la ̄
belled glycineꎬ 0􀆰 75% sucroseꎬ and 0􀆰 75% agar.
Before the experiments beganꎬ the seedlings were
3233期    WANG Qiu ̄Xia et al.: Mycorrhizal Fungi Promote Growth and Nitrogen Utilization by Dendrobium nobile 􀆺    
trimmed to provide uniformly sized materials. Three
inoculation treatments were established ( S1ꎬ S3ꎬ
and control)ꎬ with each applied as described for our
inoculation experiment. A mycelia plug was placed
near the roots of each seedlingꎬ so that suitable in ̄
fections would occur as soon as possible. Replicate
treatments (n= 6) were performed to compensate for
any contamination. The experiments were carried out
in the greenhouse (12 h photoperiodꎬ 50 μmol􀅰m-2􀅰
s-1ꎬ 26±1 ℃) for two months.
1􀆰 7  Analysis of 15N stable isotope
After two monthsꎬ the plants were harvested
from each bottle and dried at 80 ℃ for 48 h. They
were then ground to a fine powder to obtain a repre ̄
sentative subsample of tissue for further analysis. U ̄
sing the protocol of Liebel and Gebauer (2011)ꎬ we
measured the relative abundances of the N isotope
with an isotope ratio mass spectrometer ( Delta V
Advantageꎻ Thermo Fisher Scientificꎬ Inc.ꎬ USA)
at the Institute of Desertification Studiesꎬ China A ̄
cademy of Forestry. Three test substances of varying
sample weight were routinely analyzed within each
treatment. The maximum variation in δ15 N was al ̄
ways below 0􀆰 2‰.
1􀆰 8  Determination of dendrobine percentages
We extracted and quantified the levels of den ̄
drobine according to the method of Li et al. (2009)ꎬ
with some modifications. Brieflyꎬ approximately 0􀆰 3 ̄
g dry samples of D􀆰 nobile from each treatment were
soaked for 30 min in 2 mL of ammonium hydroxide.
Afterwardꎬ 50 mL of chloroform was added. The
samples were weighed and then extracted refluently
for 3 h. An appropriate amount of chloroform was
added to each sample to compensate for any weight
loss. The filtrate was evaporated under vacuum. After
the residue was dissolved with methanolꎬ it was
transferred to a 1 mL volumetric flask and diluted to
constant volume with methanol. The liquid was pas ̄
sed through a 0􀆰 45 μm microfiltration membraneꎬ
and the dendrobine content of the filtrate was deter ̄
mined via liquid chromatography ̄mass spectrometry
(LC ̄MS). The LC separation was conducted on a
Zorbax SB column (4􀆰 6 mm×250 mm i􀆰 d.ꎬ 5􀆰 0 μm).
The elution comprised 25% acetonitrile and 75%
water with 0􀆰 2% formic acid at a flow rate of 1􀆰 0 mL
min-1 . In addition to an injector volume of 10 μLꎬ
the LC ̄MS conditions included capillary voltageꎬ
2 500 Vꎻ cone voltageꎬ 30 Vꎻ temperatures of desol ̄
vationꎬ 350 ℃ꎻ and gas flow rateꎬ 400 L h-1 . Peak
retention times were compared with that of the den ̄
drobine standard. For all treatmentsꎬ we used regres ̄
sion equations to calculate the dendrobine percenta ̄
ges in terms of their peak areas. Each treatment was
repeated three times.
1􀆰 9  Data collection and statistical analysis
Two months after inoculationꎬ the plants were
harvested to record their heightsꎬ main stem lengths
and diametersꎬ internodal lengths and numbers of
nodes. For each culture bottleꎬ fresh weights were
obtained for all eight plantsꎬ and the numbers of new
roots and tillers were tallied. Those plants were then
dried for 48 h at 80 ℃ before determining their dry
weights. The increment in fresh weight per bottle was
calculated as the difference in plant weight before
and after inoculation. All data analyses were per ̄
formed with the statistical package SPSS 17􀆰 0. Diff ̄
erences among treatments and control groups were
tested with a one ̄way analysis of variance ( ANO ̄
VA)ꎬ followed by tests for least significant differ ̄
ences ( LSD). Values were presented as means ±
standard errors (SE).
2  Results
2􀆰 1   Morphological characterization of mycor ̄
rhizal fungi
Colonies of isolate S1 were light ̄yellow with
concentric ringsꎬ lacked aerial hyphaeꎬ and assumed
a leathery texture within one month (Fig􀆰 1A). The
hyphae were septateꎬ and 2􀆰 5 to 5􀆰 5 μm in diame ̄
terꎬ with nearly right ̄angled branches. The bases of
those branches were constricted (Fig􀆰 1B). Ellipsoi ̄
dal monilioid cells were 4􀆰 0 to 10􀆰 5 μm by 9􀆰 0 to
12􀆰 0 μm (Fig􀆰 1C). By contrastꎬ S3 colonies were
white and presented a cottony texture with age.
423                                  植 物 分 类 与 资 源 学 报                            第 36卷
White aerial hyphae were well developed along the
submerged margins on PDA medium (Fig􀆰 1D). The
hyphae were hyalineꎬ septateꎬ 2􀆰 0 to 5􀆰 0 μm in di ̄
ameterꎬ with nearly right ̄angled branches. Those
branch bases were also constricted ( Fig􀆰 1E). Mo ̄
nilioid cells (Fig􀆰 1F) were obovoidꎬ fusiform or ir ̄
regularly ellipsoidal ( 6 - 10 μm × 8 - 22 μm). Be ̄
cause of their cultural and morphological characteris ̄
tics (Nontachaiyapoom et al.ꎬ 2010)ꎬ both isolates
were identified as being Rhizoctonia ̄like fungi.
2􀆰 2  Molecular identifications of isolates
We amplified and sequenced the rDNA ITS re ̄
gion of S1 and the mtLSU region of S3. BLAST sear ̄
ches revealed that the ITS sequence of S1 shared
high identity (96%ꎻ 602 / 625) with that of Epulo ̄
rhiza sp. ( Table 1). The mtLSU sequence of S3
shared 98% identity (316 / 321) with uncultured Tu ̄
lasnella sp. and 96% identity (317 / 330ꎻ 314 / 327)
with Tulasnella sp. (Table 2). Based on their mor ̄
phological and molecular dataꎬ we identified S1 and
S3 as Epulorhiza sp. and Tulasnella sp.ꎬ respec ̄
tively.
2􀆰 3  Visualization of fungal infection
After two months of inoculationꎬ the S1 and S3
hyphae had spread over the root surfacesꎬ invaded
the cortical cellsꎬ and colonized the root intracellular
Fig􀆰 1  Cultural and morphological characteristics of mycorrhizal fungi on PDA media. Isolate S1: Aꎬ Colony (Scale bar = 1 cm)ꎻ
Bꎬ hyphae (Scale bar = 2 μm )ꎻ Cꎬ ellipsoidal monilioid cells (Scale bar = 2 μm) Isolate S3: Dꎬ Colony
(Scale bar = 1 cm)ꎻ Eꎬ hyphae (Scale bar = 4 μm)ꎻ Fꎬ monilioid cells (Scale bar = 10 μm)
Table 1  Blast results for the S1 isolate based on its ITS region
Strain Percent identity Gap Reference
FJ594913 Epulorhiza sp. 96% (602 / 625) 2% (10 / 625) Li et al.ꎬ unpublished
FJ594914 Epulorhiza sp. 96% (601 / 624) 2% (10 / 624) Li et al.ꎬ unpublished
FJ594919 Epulorhiza sp. 96% (600 / 623) 2% (10 / 623) Li et al.ꎬ unpublished
FJ594918 Epulorhiza sp. 96% (598 / 621) 2% (10 / 621) Li et al.ꎬ unpublished
FJ594916 Epulorhiza sp. 96% (599 / 624) 2% (10 / 624) Li et al.ꎬ unpublished
FJ594915 Epulorhiza sp. 96% (594 / 617) 2% (10 / 617) Li et al.ꎬ unpublished
FJ594917 Epulorhiza sp. 96% (595 / 619) 2% (12 / 619) Li et al.ꎬ unpublished
FJ594912 Epulorhiza sp. 96% (594 / 618) 2% (11 / 618) Li et al.ꎬ unpublished
GQ241863 uncultured Tulasnellaceae 96% (597 / 620) 1% (8 / 620) Yuan et al.ꎬ 2010
5233期    WANG Qiu ̄Xia et al.: Mycorrhizal Fungi Promote Growth and Nitrogen Utilization by Dendrobium nobile 􀆺    
spaces of inoculated seedlings (Fig􀆰 2Aꎬ B). In the
cortical regionꎬ the hyphae penetrated through the
cell walls and entered next to the cortical cells
(Fig􀆰 2C). Under the LSCMꎬ no fungal hyphae were
observed within root cells from the control ( non ̄in ̄
oculated) seedlings (Fig􀆰 2D).
Table 2  Blast results for the S3 isolate based on its mtLSU region
Strain Percent identity Gap Reference
AY192522 uncultured Tulasnella sp. 98% (316 / 321) 0 (0 / 321) Bidartondo et al.ꎬ 2003
AY192520 uncultured Tulasnella sp. 98% (316 / 321) 0 (0 / 321) Bidartondo et al.ꎬ 2003
AY192514 uncultured Tulasnella sp. 98% (316 / 321) 0 (0 / 321) Bidartondo et al.ꎬ 2003
AY192512 uncultured Tulasnella sp. 98% (316 / 321) 0 (0 / 321) Bidartondo et al.ꎬ 2003
AY192511 uncultured Tulasnella sp. 98% (316 / 321) 0 (0 / 321) Bidartondo et al.ꎬ 2003
AY382811 Tulasnella pruinosa 98% (318 / 326) 0 (1 / 326) McCormick et alꎬ 2004
AF345560 Tulasnella irreqularis 98% (312 / 320) 0 (1 / 320) Kristiansen et al.ꎬ 2001
AY382794 Tulasnella sp. 96% (317 / 330) 1% (2 / 330) McCormick et al.ꎬ 2004
DQ834411 Tulasnella sp. 96% (314 / 327) 1% (2 / 327) Porras ̄Alfaro and Baymanꎬ 2007
Fig􀆰 2  Fungal infection in Dendrobium nobile seedlings. A. Inoculation with S1ꎻ hyphae and hyphal coils (green) in root cortical cellsꎻ
B. Inoculation with S3ꎻ hyphae and hyphal coils (green) in root cortical cellsꎻ C. Inoculated with S1ꎻ hyphae (green) penetrating
cell wall and invading neighboring cells in rootꎻ D. Root cortical cells from non ̄inoculated control seedlings. Scale bar = 50 μm
623                                  植 物 分 类 与 资 源 学 报                            第 36卷
2􀆰 4  Growth responses by D􀆰 nobile to fungal in ̄
oculation
At two months after inoculationꎬ growth was en ̄
hanced for the host plants (Table 3). Compared with
the untreated controlꎬ increment of fresh weights and
internodal lengths were significantly increased for
seedlings inoculated with either S1 or S3 fungi. How ̄
everꎬ the numbers of nodes were not significantly dif ̄
ferent among control and treated plants. Dry weightsꎬ
stem lengths and diameters and numbers of tillers
were significantly higher for S1 ̄ inoculated seedlings
than for the control. Howeverꎬ plant heights and num ̄
bers of new roots did not differ significantly between
the two types. When the control and S3 ̄inoculated
seedlings were comparedꎬ no significant differences
were found with dry weightsꎬ stem lengths and diame ̄
tersꎬ and numbers of tillers. Values for all growth
characteristicsꎬ except plant heights and numbers of
nodesꎬ were significantly higher for S1 ̄ inoculated
seedlings than for S3 ̄ inoculated seedlings.
The S1 and S3 fungi were re ̄isolated from the
D􀆰 nobile roots at two months post ̄inoculation. Their
cultural and morphological characteristics were the
same as those recorded for the original isolates. We
also sequenced their ITS ̄5􀆰 8S rDNA sequences and
mtLSU sequences from the inoculated rootsꎬ and
verified that the re ̄isolated fungi were the same as
those used for the first inoculations.
2􀆰 5  Nitrogen utilization by different treatments
The15N derived from 15N ̄labelled glycine was
readily detectable in the inoculated and non ̄inocula ̄
ted seedlings (Fig􀆰 3). Although the δ15N value in
the S1 ̄ inoculated seedlings was significantly higher
than that in the control and S3 ̄inoculated seedlingsꎬ
that value did not differ significantly between the
control and S3 ̄ inoculated seedlings.
2􀆰 6  Dendrobine contents
Infection with mycorrhizal fungi considerably
increased the levels of dendrobine by 60 d post ̄inoc ̄
ulation (Fig􀆰 4). Contents were about 25 ̄ (S1) and
2 ̄ fold (S3) higher than in the control seedlings. In
additionꎬ the percentage of dendrobine was signifi ̄
cantly higher in S1 ̄inoculated seedlings than in
those exposed to the S3 isolate.
3  Discussion
Both S1 and S3 isolates were identified as being
Rhizoctonia ̄like fungi. Because mycelia from that
type do not yield many distinguishing charactersꎬ i ̄
dentification is generally not possible below the ge ̄
neric level (Rasmussenꎬ 2002). Thusꎬ we used two
genes to aid in our examination. Based on the se ̄
quences in the NCBI database and effective primers
(Bruns et al.ꎬ 1998ꎻ Ma et al.ꎬ 2003) for their
ITS ̄5􀆰 8S rDNA and the mtLSU rDNA sequencesꎬ
we were able to identify S1 and S3 as being species
within Epulorhiza and Tulasnellaꎬ respectively. These
have previously been reported as mycorrhizal fungi of
orchid plants (Ma et al.ꎬ 2003ꎻ Nontachaiyapoom et
al.ꎬ 2010).
Table 3  Influence of S1 and S3 fungal isolates on the growth of Dendrobium nobile seedlings (n= 25)
Parameter S1 S3 Control
Increment of fresh weight / g 0􀆰 88 ± 0􀆰 06b 0􀆰 72 ± 0􀆰 05c 0􀆰 50 ± 0􀆰 03a
Dry weight / g 0􀆰 08 ± 0􀆰 00b 0􀆰 04 ± 0􀆰 00a 0􀆰 03 ± 0􀆰 00a
Plant height / cm 2􀆰 37 ± 0􀆰 06ab 2􀆰 10 ± 0􀆰 08b 2􀆰 54 ± 0􀆰 14a
Stem length / cm 1􀆰 29 ± 0􀆰 04b 1􀆰 08 ± 0􀆰 05a 1􀆰 03 ± 0􀆰 03a
Stem diameter / cm 0􀆰 21 ± 0􀆰 01b 0􀆰 18 ± 0􀆰 01a 0􀆰 16 ± 0􀆰 01a
Number of nodes 3􀆰 33 ± 0􀆰 12a 3􀆰 16 ± 0􀆰 13a 3􀆰 23 ± 0􀆰 09a
Internodal length / cm 0􀆰 36 ± 0􀆰 01b 0􀆰 32 ± 0􀆰 01c 0􀆰 28 ± 0􀆰 01a
Number of tillers 17􀆰 28 ± 0􀆰 79b 12􀆰 65 ± 0􀆰 76a 11􀆰 00 ± 0􀆰 81a
Number of new roots 22􀆰 84 ± 1􀆰 60a 17􀆰 15 ± 1􀆰 05b 22􀆰 80 ± 1􀆰 48a
Different letters within the same row indicate that values (mean±standard error) are significantly different among treatments at P<0􀆰 05ꎬ based on
LSD tests. Controlꎬ non ̄inoculated seedlings
7233期    WANG Qiu ̄Xia et al.: Mycorrhizal Fungi Promote Growth and Nitrogen Utilization by Dendrobium nobile 􀆺    
Fig􀆰 3  δ15N calculated from inoculated and non ̄inoculated
(control) seedlings of Dendrobium nobile (n= 3). Values
(mean ± standard error) not followed by the same letter
are significantly different at P<0􀆰 05ꎬ
based on LSD tests
Fig􀆰 4  Effects of mycorrhizal fungi treatment on dendrobine
contents in Dendrobium nobile seedlings (n= 3) Values
(mean ± standard error) not followed by the same letter
are significantly different at P<0􀆰 05ꎬ based on
LSD tests. Controlꎬ non ̄inoculated seedlings
    Although Epulorhiza sp. is known to stimulate
biomass accumulations by plants of D􀆰 nobile (Song
and Guoꎬ 2001ꎻ Chen and Guoꎬ 2005)ꎬ the effects
of Tulasnella sp. on Dendrobium have rarely been
described ( Nontachaiyapoom et al.ꎬ 2011). Some
earlier studies have demonstrated that Tulasnella sp.
promotes seed germination and growth by Vanilla sp.
and Pecteilis susannae (Porras ̄Alfaro and Baymanꎬ
2007ꎻ Chutima et al.ꎬ 2011). Our fingdings indica ̄
ted that both S1 and S3 successfully colonized the
roots of D􀆰 nobile and formed some mycorrhizal struc ̄
tures. Moreoverꎬ both fungi had positive influences
on the growth of their host plants.
The S1 ̄ inoculated seedlings utilized signifi ̄
cantly more organic N from amino acids in compari ̄
son with the control seedlings. Howeverꎬ the number
of newly initiated roots was not significantly different
between the two. Thusꎬ most of the measured uptake
of amino acids was probably by the fungi rather than
the roots ( Jones et al.ꎬ 2005). We also deduced
here that more of the acquired organic N was trans ̄
ferred to the seedlings by the fungi. By contrastꎬ δ15
N values did not differ significantly between the S3  ̄
inoculated and control seedlings. This suggested that
S1 and S3 fungi have different nutrient roles within
the same host. Similarlyꎬ Midgley et al. ( 2006)
have shown that the ability of a host plant to access
nutrients from organic substrates depends upon the
capacity of its associated fungi.
Both S1 ̄ and S3 ̄ inoculated seedlings per ̄
formed better than the controlꎬ demonstrating that N ̄
availability is of great importance. The supply of ni ̄
trogen has very marked effects on stem and leaf
growthꎬ and number of tillers. This is manifested by
higher leaf ̄N contents and larger leaf areas. Up to
75% of the leaf N is found in the chloroplastsꎬ with
most of that being invested in the production of ribu ̄
lose bisphosphate carboxylase alone ( Cechin and
Fumisꎬ 2004). Consequentlyꎬ increasing the supply
of N improves chlorophyll contents and Rubisco ac ̄
tivityꎬ thereby enhancing the rates of photosynthesis
and biomass accumulation. Our data showed that
considerably more organic N was acquired by the S1 ̄
inoculated seedlings than by the control. Thusꎬ the
higher rate of photosynthesis was linked with the pro ̄
motion of plant growth. An abundant N supply in ̄
creases the number of meristems and the formation of
shoots (Lawlor et al.ꎬ 1989). Hereꎬ we investigated
only the acquisition of organic N. Howeverꎬ inorga ̄
nic ̄N nutrition is also criticalꎬ being obtained by the
plants directly or else transferred through symbiotic
fungi (Gebauer and Meyerꎬ 2003). Thusꎬ we be ̄
823                                  植 物 分 类 与 资 源 学 报                            第 36卷
lieve that the growth of our S3 ̄inoculated seedlings
was possibly improved because more non ̄isotopically
labelled inorganic N was acquired.
Our results also indicated that dendrobine con ̄
tents were significantly increased in seedlings inocu ̄
lated with either the S1 or S3 isolates. Chen and Guo
(2005) have also reported that the total alkaloid
content can rise by 18􀆰 3% when D. nobile seedlings
are co ̄cultured with Mycena. Howeverꎬ earlier in ̄
vestigations have not focused on dendrobineꎬ a main
bioactive component. Hereꎬ dendrobine contents
were significantly elevated within infected seedlings
when compared with the control. This suggestedꎬ
thereforeꎬ that the quality of tissue ̄cultured seed ̄
lings might be elevated by inoculation with mycorrhi ̄
zal fungi. Although all mineral nutrients can affect
the composition of plant tissues to some extentꎬ N is
particularly influential. Its increased availability can
lead to greater concentrations of nitrogen ̄containing
compoundsꎬ such as alkaloids (Johnson et al.ꎬ 1987).
Similarlyꎬ we could conclude that the rise in den ̄
drobine contents in the inoculated seedlings was due
to better acquisition of organic and / or inorganic N
by the symbiotic fungi.
In summaryꎬ our data demonstrate that both S1
and S3 isolates form mycorrhizal associations with
D􀆰 nobile. Moreoverꎬ seedling growthꎬ organic ̄N ac ̄
quisitionsꎬ and dendrobine accumulations can be
promoted through these infections. Neverthelessꎬ al ̄
though those fungi have been proven beneficial to
this photosyntheticꎬ epiphytic orchid under laborato ̄
ry conditionsꎬ further examinations are necessary to
determine whether this symbiotic relationship can al ̄
so influence plants of this species in the field. We
must also continue to study the mechanism by which
dendrobine contents are increased in inoculated
plants.
Acknowledgements: The authors are grateful to Dr. Jinsong
Wu for his guidance in the visualization of fungal infections by
laser scanning confocal microscopyꎻ We also thank the Germ ̄
plasm Bank of Wild Species in Southwest Chinaꎬ for provi ̄
ding access to that microscope.
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