全 文 ：
菌物学报
jwxt@im.ac.cn 22 February 2017, 36(2): 1‐13
Http://journals.im.ac.cn Mycosystema ISSN1672‐6472 CN11‐5180/Q
Tel: +86‐10‐64807521 Copyright © 2016 Institute of Microbiology, CAS. All rights reserved.
研究论文 Research paper DOI: 10.13346/j.mycosystema.160018
Supported by the National Natural Science Foundation of China (31470101, 31272210) and the Basic Research Project of
Qingdao Government [12‐1‐4‐5‐(14)‐jch].
*Corresponding author. E‐mail: liurj@qau.edu.cn
Received: 2016‐01‐18, accepted: 2016‐09‐02
Diversity of arbuscular mycorrhizal fungi and dark septate endophytes in
the greenhouse cucumber roots and soil
HU Yu‐Jin1 GAO Chun‐Mei1 LIU Xing‐Zhong2 LIU Run‐Jin1*
1Institute of Mycorrhizal Biotechnology, Qingdao Agricultural University, Qingdao 266109, China
2State Key Laboratory of Mycology, Institute of Microbiology, Chinese Academy of Sciences, Beijing 100101, China
Abstract: The diversity and its ecological functions of arbuscular mycorrhizal fungi (AMF) and dark septate endophytes (DSE) on
wild plants have been well documented. However, the species of AMF and DSE and their function simultaneously colonizing on
the same root system of cultivated crops are less investigated. The purpose of the present study was to monitor AMF and DSE in
the roots of cucumber (Cucumis sativus Linn.) and soils in the greenhouse condition by using traditional morphological methods
and nested polymerase chain reaction‐denaturing gradient gel electrophoresis (PCR‐DGGE). For the AMF on cucumber roots, 7
species including Glomus fasciculatum, Glomus indicum, Funneliformis mosseae, Scutellospora dipurpurescens, Gigaspora
margarita and two uncultured species of Archaeospora were detected with PCR‐DGGE, while only F. mosseae, G. indicum and Gi.
margarita were detected based on the morphological identification of spores collected from the root trap culture. Meanwhile, 6
isolates of DSE were isolated from the cucumber root segments and one of them was identified as Phoma leveillei with
PCR‐DGGE. Twenty species in 9 genera of AMF were isolated and identified based on spores extracted in the cucumber root zone
soil. Glomus was the dominant genus in cucumber roots under greenhouse conditions. More AMF species in cucumber roots
were detected by molecular techniques in comparison with the species detected by the trap culture and the spore
morphological methods.
Key words: arbuscular mycorrhizal fungi, dark septate endophytes, protected agricultural soil, Glomus, Phoma leveillei,
cucumber roots
网络出版时间：2016-10-31 15:36:30
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ISSN1672‐6472 CN11‐5180/Q Mycosystema February 22, 2017 Vol. 36 No. 2
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保护地黄瓜根内和土壤中丛枝菌根真菌和深色有隔内生真菌
多样性研究
胡玉金 1 高春梅 1 刘杏忠 2 刘润进 1*
1青岛农业大学菌根生物技术研究所 山东 青岛 266109
2中国科学院微生物所真菌学国家重点实验室 北京 100101
摘 要：对于野生植物根内定殖的丛枝菌根真菌（AMF）和暗隔内生真菌（DSE）多样性及其生态功能现已进行了众多的
调查研究。然而，关于这两种真菌同时定殖于栽培作物的同一根系的物种多样性和功能我们却了解甚少。本研究旨在采
用传统的形态学方法和 PCR‐DGGE 技术探究保护地栽培的黄瓜 Cucumis sativus Linn.根内 AMF 和 DSE 的物种多样性。
PCR‐DGGE 结果显示共有 7 种 AMF，包括 Funneliformis mosseae，Glomus fasciculatum，Glomus indicum，Scutellospora
dipurpurescens，Gigaspora margarita 以及两个未培养的 Archaeospora；而以黄瓜植株根段作为接种物加富培养后，依据其
所产生的孢子形态特征进行分类鉴定，则只分离获得 3 种，即 F. mosseae，G. indicum 和 Gi. Margarita；同时，采用常规
分离纯化的方法从黄瓜根内分离得到 DSE 6 个菌株，其中 1 株经分子生物学鉴定为 Phoma leveillei。从保护地根区土壤中
分离 AMF 孢子并通过形态学分类鉴定，获得了 9 属 20 种。研究结果表明，Glomus 是保护地栽培黄瓜根系内的优势属，
针对数量，相对于传统形态鉴定技术，分子技术可以检测到根内更多的 AMF。
关键词：丛枝菌根真菌，深色有隔内生真菌，农业保护地土壤，黄瓜根系
INTRODUCTION
Symbiosis is an intimate association between
two different organisms through all or part of their
life cycle. The continued development of ecology,
powered by advances in molecular identification
methods has revealed the widespread occurrence
and identity of symbiotic relationships with
microbes in many groups of organisms (Kurtzman &
Fell 2006). Although symbioses between plant roots
and bacteria, such as plants with nitrogen‐fixing
Rhizobia (Sprent & James 2007) were considered
primitive associations, arbuscular mycorrhizal fungi
(AMF) and dark septate endophytes (DSE) are
probably the most widespread and dominant
symbioses between fungi and plant roots (Baslam et
al. 2012) when plants are well established in
terrestrial ecosystems. The establishment of these
symbioses has been proposed to be a key event that
allowed some primitive plants to colonize the harsh
land environment for the first time. The AM
symbiosis is one of the most fundamental keys to
understanding many processes in plant and soil
biology and ecology (Abbott & Lumley 2014).
AMF are distributed widely in farmland, forest,
desert, saline‐sodic soil and greenhouse soils (Liu et
al. 2014). AMF promote plant growth by improving
mineral nutrition and water status of plants,
increase plant resistance to pathogens and
tolerance to the other biotic and abiotic stresses (Liu
et al. 2012; Wu et al. 2012; Xie et al. 2014).
Dark septate endophytes (DSE) are a group of
endophytic fungi characterized by their morphology
of melanized septate hyphae and colonize roots
HU Yu‐Jin et al /Diversity of arbuscular mycorrhizal fungi and dark septate endophytes in the greenhouse cucumber roots and soil
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intracellularly or intercellularly (Sieber & Grünig
2006). DSE are ubiquitous in various stressful
environmental conditions, interrelated with many
plants ranging from the tropics to the arctic and are
particularly common in habitats under stress such as
alpine environments. DSE fungi, like mycorrhizal
fungi, exhibit a very broad host range and can
colonize nearly 600 plant species in about 320
genera and 114 families (Jumpponen & Trappe
1998). DSE often enhance plant growth by
improving nutrient uptake, increasing tolerance
against root pathogens, strengthening ability to
withstand adverse environmental conditions (Wu et
al. 2010).
Plant roots encounter both AMF and DSE under
natural conditions (Saravesi et al. 2014), and both of
them can infect plant roots to form dual symbioses
(García et al. 2012; Liu et al. 2014). DSE can colonize
mycorrhizal plant under natural conditions
(Massenssini et al. 2014) to span the continuum
from mildly antagonistic to mutualistic interactions
(Jumpponen 2001) on vegetation under natural
conditions, especially on wild plants (Massenssini et
al. 2014). However, in comparison with wild plants,
we know little about the simultaneous colonization
by AMF and DSE on crop roots. An understanding of
the colonization of crop roots by both AMF and DSE
and their species diversity will provide insight for
the function of AMF and DSE in agriculture system.
Many species of AMF have been isolated and
identified from vegetable crops grown in
greenhouse in north China and in natural grassland
(Jiao et al. 2011; Kim et al. 2014). During our
previous work, fungal structures of both AMF and
DSE have been observed simultaneously in one root
of cucumber grown in greenhouse soils (Tian et al.
2015). Soil is the base camp of microbial life, as well
as microbial resources. The native species diversity
of AMF and DSE in soil provides resources for the
root colonization of the fungi. The purpose of the
present study was to determine the species diversity
of AMF and DSE in the root zone soil and the roots
of cucumber grown in the greenhouse.
1 MATERIALS AND METHODS
1.1 Sampling sites and the sample treatment
Three greenhouses with cucumber
monoculture for more than five years were selected
in Laiyang (120°71′ E and 36°99′ N), Laixi
(120°52′ E and 36°93′ N) and Shouguang
(120°26′ E and 36°45 N), the region of greenhouse
cucumber production in Shandong. The sampling
was conducted at the different development stages
of cucumber plants including stages of seedling,
flowering, initial fruit setting (50 days after
transplantation), full fruit period (70 days after
transplantation), and preharvest period (90 days
after transplantation) (Wang et al. 2012) in the
growing season of 2013. Five sampling plots in each
greenhouse were randomly selected, and five plants
in each plot were collected and ca. 1 000g soil
around the sampling plant roots by borer in 0–30cm
depth with roots were taken and placed in a sealed
plastic bag. Part of soil was used to isolate AMF
spores for species identification. Root samples were
separated into two parts except for the sample
collected in the last fruiting stage which was
separated into 3 parts. One part was stored under
‐80℃ for DNA extraction, the other part of the
fresh roots was employed to isolate DSE. For precise
identification to enrich AMF spores, a trap culture
trial (Liu & Chen 2007) was conducted for the root
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and soil samples of the last fruiting stage. The
trapping plants, clover (Trifolium subterraneum) and
tobacco (Nicotiana tabacum), were planted in the
pots that contain the soil sample or the autoclaved
soil inoculated with the root sample that was cut
into 0.3–0.5cm length segments. The root zone soil
samples collected in the last stage were also
enriched in trap culture to isolate AMF.
1.2 Species diversity of arbuscular mycorrhizal fungi
After two month cultivation, AMF spores were
extracted from the trap culture soils by wet sieving
(20–400μm mesh) and decanting, followed by
sucrose density centrifugation (Liu & Chen 2007).
AMF species identification was based on spore
morphology, original descriptions of species, and
detailed descriptions provided by the International
Collection of Vesicular and Arbuscular Mycorrhizal
Fungi (http://invam.caf.wvu.edu) and Polish
Agricultural University Professor Janusz Blaszkowski
(http://www.zor.zut.edu.pl/ Glomeromycota/).
Spore density in the root zone soil of the
greenhouse was expressed as the number of spores
in 100g air‐dried soil, and species richness was the
number of AMF taxa detected, Shannon‐Weiner
index was calculated according to the following
formula:
H´=－∑(Pi)ln(Pi)
Where Pi= ni/N, ni represents the number of spores
for an individual species, and N represents total
number of spores.
1.3 Isolation of dark septate endophytes
Roots of the cucumber plants were washed in
running tap water and surface‐sterilized with 75%
ethanol for 5min, and then 10% sodium hypochlorite
for another 5min. The surface‐sterilized roots were
cut into small pieces (ca. 5mm) and placed on 90mm
Petri dishes containing potato dextrose agar (PDA).
Three to five root segments were placed onto each
PDA plate and incubated in the dark for about 30d
at 24℃. The plates were daily observed and the
dark mycelium growing from the root segments
were transferred onto new PDA plates for
purification.
1.4 Molecular identification of arbuscular
mycorrhizal fungi and dark septate endophytes
1.4.1 DNA extraction: Approximately 20mg of each
root sample collected from greenhouse and 2‐ DSE
culture after incubation for two weeks were used
for AMF and DSE DNA extraction respectively. The
root samples were cut into 1‐2mm fragments and
ground in liquid nitrogen. The total DNA was
extracted using the E.Z.N.A.® Plant DNA Miniprep kit
(Omega Bio‐Tek, Inc., Norcross, GA, USA) according
to the manufacturer’s protocol, while DSE DNA was
extracted by the CTAB method (Grünig et al. 2002).
Each DNA sample was finally dissolved in 100μL
elution buffer and stored at ‐20℃ until further
assay.
1.4.2 Nested PCR and DGGE analysis: (1): DSE
Primers ITS1 (5‐TCCGTAGGTGAACCTGCGC‐3) and
ITS4 (5‐TCCTCCGCTTATTGATATGC‐3) were
employed to amplify DSE fungal rDNA internal
transcribed spacer (ITS). The PCR reactions were
performed in a S1000 Thermal Cycler (Bio‐Rad, USA)
and 50μL reaction volume containing 2μL genomic
DNA, 2μL of each primer (10mmol/L, 5μL 10× PCR
buffer, 7μL 25mmol/L Mg2+, 2μL 2.5mmol/L dNTP,
1μL 5U/μL Taq DNA polymerase, and 29μL ddH2O
was applied. The conditions included an initial
denaturation at 94℃ for 3min, followed by 35
cycles at 94℃ for 1min, 55℃ for 45s, and 72℃ for
2min, and a final extension at 72℃ for 8min. PCR
HU Yu‐Jin et al /Diversity of arbuscular mycorrhizal fungi and dark septate endophytes in the greenhouse cucumber roots and soil
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products were analyzed with 1.2% (W/V) agarose gel
electrophoresis (130V, 20min) and stained with
ethidium bromide, and bands were visualized under
UV light. Expected bands were excised and purified
with E.Z.N.A.H Gel extraction kit (Omega Bio‐Tek,
Inc., Norcross, GA, USA). PCR amplification products
were sequenced by Shanghai Biotechnology Co., Ltd.
All DNA sequences were edited and compared to
the available sequences from the National Center
for Biotechnology Information (NCBI) using the basic
local alignment search tool (BLAST).
(2) AMF: The DNA sample extracted from the
root was subjected to a first PCR amplification using
primers GeoA2 and Geo11 to amplify an
approximately 1.8kb fragment of the 18S rRNA gene
(Schwarzott & Schüβler 2001). The first‐round
products were diluted to 1/100 and used as
templates in the second‐round PCR, using primers
AM1 (Helgason et al. 1998) and NS31‐GC (Simon et
al. 1992). The second‐round primers produced an
approximately 550bp fragment. The products were
diluted to 1/100 to be used as templates in the
third‐round PCR, using primers NS31‐GC and Glo1
(Cornejo et al. 2004). The third‐round PCR produced
a DNA fragment of about 250bp. All PCR
amplifications were performed in a final volume of
25μL mixture containing 2.5μL 10× PCR buffer, 1μL
2.5mmol/L dNTP mix, 0.5μL of each primer
(10mmol/L), 0.1μL 5U/μL Taq DNA polymerase, 1μL
genomic DNA and 19.4μL ddH2O. PCR was
conducted as follows: for the first round, 94℃ for
4min; 30× (94℃, 1min; 54℃, 1min; 72℃, 2min);
72℃, 7min; for the second round, 94℃ for 2min;
30× (94℃, 45s; 65℃, 1min; 72℃, 45s); 72℃, 7min;
for the third round, 94℃ for 4min; 30× (94℃, 45s;
55℃, 1min; 72℃, 45s); 72℃, 7min. PCR products
were analyzed by 1.2% (W/V) agarose gel
electrophoresis (130V, 20min) and stored at −20℃
for subsequent analysis.
(3) DGGE analysis: The PCR‐DGGE was
performed with a D‐Code Universal Mutation
Detection System (Bio‐Rad, Hercules, CA, USA). Gels
contained 8% (W/V) polyacrylamide (37.5:1
acrylamide/ bis‐acrylamide) and 1×TAE (Tris/acetic
acid/EDTA) buffer. The used linear gradient was
from 20% to 55% denaturant, where 100%
denaturing acrylamide was defined as containing
7mol/L urea and 40% (V/V) formamide.
Electrophoresis was conducted for 10min at 120V,
after which the voltage was lowered to 80V for an
additional 12h in 1× TAE buffer at a constant
temperature of 60℃ . Gels were stained with
ethidium bromide and visualized under ultraviolet
light using a Gel Doc XR documentation system
(Bio‐Rad, Hercules, CA, USA), and the intensity of
bands was analyzed by Quantity One software
(Bio‐Rad, Hercules, CA, USA). The analysis of AMF
diversity indices was performed based on the band
number, position, and intensity in the DGGE profiles.
(4) Phylogenetic analysis: For the construction
of the phylogenetic tree of DSE, sequence alignment
was carried out using ClustalW, and phylogenetic
analyses were conducted with MEGA 5.0 (Tamura et
al. 2011) using the neighbor‐joining method with
the Kimura two‐parameter distance measure.
Confidence values were estimated from bootstrap
analysis of 1 000 replicates.
For AMF identification, single dominating bands
were excised from the DGGE gel (Kowalchuk et al.
2002; Jiao et al. 2011), the purified PCR products
were cloned by ligation with pGEM‐T Easy vector (de
Souza et al. 2004). All DNA sequences were edited
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and compared to the available sequences of NCBI by
using BLAST. The neighbor‐joining tree was
constructed using Tamura‐Nei nucleotide
substitution model (in MEGA 5.0）(Tamura et al.
2011).
1.5 Statistical analysis
The data of species richness, spore density and
Shannon‐Weiner index were analyzed by one‐way
analysis of variance (ANOVA) using SPSS 16.0
software (SPSS Inc., Chicago, IL). Banding patterns of
the DGGE profiles were processed using Quantity
One software (Bio‐Rad, USA). All DNA sequences
were edited and compared to the available
sequences from NCBI using BLAST.
2 RESULTS
2.1 Species of arbuscular mycorrhizal fungi in
cucumber roots and the root zone soil
There were only Funneliformis mosseae,
Glomus indicum and Gigaspora margarita identified
according to morphological characteristics of spores
isolated from the trap culture that was inoculated
with the surface sterilized root segments of the
cucumber plants collected in the fruiting stage,
while 20 species were identified from the trap
culture with native root zone soil of cucumber
plants. This result indicated that the soils have more
AMF taxa and only a few AMF species can infect the
cucumber roots. Among them, eight species in
Glomus, three in Scutellospora, each two in
Ambispora and Rhizophagus, each one in
Acaulospora, Claroideoglomus, Funneliformis,
Gigaspora and Sclerocystis. Glomus widely
distributed at all three sampling sites. Scutellospora
was detected in the samples collected at Laiyang
and Laixi, Acaulospora and Claroideoglomus in
Shouguang, and Gigaspora was only detected in
Laixi (Table 1).
Both morphological method and DGGE analysis
resulted in the similar trends of the AFM diversity,
e.g. the diversity was the highest at the full fruiting
stage, moderate at the first fruiting stage and less at
the flowering stage. The species richness and spore
density were higher in Laiyang than that in Laixi and
Shouguang (Table 2, 3; Fig. 1).
Table 1 Species of arbuscular mycorrhizal fungi isolated in
root zone soil from three sites in Shandong, China
AMF species Laiyang Laixi Shouguang
Acaulospora laevis ‐ ‐ +
Ambispora callosa + + ‐
A.leptotichum + + +
Claroideoglomus
etunicatum
‐ ‐ +
Funneliformis mosseae + + +
Gigaspora margarita ‐ + ‐
Glomus aggregatum ‐ + +
G. claroideum ‐ + ‐
G. convolutum + ‐ ‐
G. indicum + + +
G. melanosporum + + ‐
G. microcarpum ‐ ‐ +
G. pansihalos + + ‐
G. reticulatum + + +
Rhizophagus clarus + + ‐
R. fasciculatus + + +
Sclerocystis clavispora + ‐ +
Scutellospora
dipurpurescens
+ ‐ ‐
S. nigra + + ‐
S. reticulata + ‐ ‐
Note: “+”, the isolate was obtained; “‐”, the isolate was not
HU Yu‐Jin et al /Diversity of arbuscular mycorrhizal fungi and dark septate endophytes in the greenhouse cucumber roots and soil
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obtained.
Table 2 Species richness and biodiversity of arbuscular mycorrhizal fungi in root zone soil of cucumber plants at various
growth stages
Developmental stage of
cucumber plants
Species
richness
Spore density
(Numbers/100g soil)
Shannon‐Weiner index
(H)
Seedling stage 1.2±0.8e 8.2±2.9d 1.08bc
Flowering stage 3.0±0.6cd 21.9±14.5c 1.04c
Initial fruiting stage 4.1±0.6c 79.3±55.8b 1.15b
Full fruiting stage 7.8±1.0b 147.5±74.6a 1.34a
Last fruiting stage 10.1±0.4a 134.2±54.1a 1.27ab
Note: Different lowercase letters represent significant difference at P<0.05.
Table 3 Species richness, spore density and biodiversity of arbuscular mycorrhizal fungi in cucumber root zone soil of the
greenhouse located in three sites
Sites Species richness Spore density (Numbers/100g soil) Shannon‐Weiner index (H)
Laixi 6.3±0.5b 118.3±5.8b 1.55a
Laiyang 7.9±0.4a 147.1±41.8a 1.49a
Shouguang 6.0±0.7b 100.7±8.6b 1.05b
Note: Different lowercase letters represent significant difference at P<0.05.
A total of 7 bands from the amplified 18S rDNA
fragments of AMF in cucumber roots were excised,
cloned, and sequenced. The results of BLAST
showed that all the sequences had high similarity to
the sequences from members of Glomeromycota.
Based on the closest BLAST matches and
phylogenetic analysis (Fig. 1), it was obvious that
Glomus was the predominant AMF genus in the
cucumber roots. In Fig. 1, bands 3, 4, 5, 6 and 7
were corresponding to Scutellospora dipurpurescens,
Glomus mosseae, G. fasciculatum, G. indicum and
Gigaspora margarita respectively. Other sequences
were identified as uncultured Archaeospora (Fig. 2).
Total 20 species of AMF were identified with
trap culture of the native cucumber root zone soils
by their morphological approaches, indicated the
richness of AMF in the soils. Compared with 3
species identified with the trap culture inoculated
with cucumber roots, 7 species of AMF were
detected by DGGE, indicated the molecular
approach is more sensitive than the morphological
method.
Ⅰ Ⅱ Ⅲ Ⅳ Ⅴ Ⅵ Ⅶ Ⅷ
2.2 Species of dark septate endophytes in the
cucumber roots
Melanized septate hyphae were observed in
the root cortex. A total of six isolates F2‐1‐3, F2‐1‐7,
F2‐2‐2, F2‐2‐5, F2‐2‐9 and F2‐3‐10 of DSE were
isolated from the sample roots of cucumber plants.
The colony of F2‐1‐3 was villous, cottony, shaggy,
gray green, deep color edge, with forming a
ring‐shaped fold. The colony of F2‐1‐7 was dark
gray‐green, velvet‐like, compact, intermediate
projections, neat edge, while F2‐2‐2 was light gray,
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with fuzzy edge. A dark green and unclear edge
colony with relatively thin mycelia attached to the
Fig. 1 DGGE pattern of nested‐PCR amplified 18S rDNA fragments of arbuscular mycorrhizal fungi in the cucumber roots. The
linear gradient used was from 30% to 60% denaturant. The bands labeled 1–7 were selected and excised from the DGGE gels and
used for cloning and sequencing. Ⅰ: In full fruiting stage at Laiyang; Ⅱ: In full fruiting stage at Laixi; Ⅲ: In full fruiting stage at
Shouguang; Ⅳ: In seedling stage at Laiyang; Ⅴ: In flowering stage at Laiyang; Ⅵ: In initial fruiting stage at Laiyang; Ⅶ: In full
fruiting stage at Laiyang; Ⅷ: In last fruiting stage at Laiyang.
HU Yu‐Jin et al /Diversity of arbuscular mycorrhizal fungi and dark septate endophytes in the greenhouse cucumber roots and soil
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Fig. 2 Neighbor‐joining phylogenetic tree inferred from partial 18S rDNA sequences of all identified arbuscular mycorrhizal fungi
in the cucumber roots.
surface of the culture medium was observed on the
PDA culture of F2‐2‐5. A cotton wool, grayish white,
gradually become gray, with deep color on the back
was showed during the culture of F2‐2‐9. The isolate
F2‐3‐10 produced dark colony on PDA plates and the
hyphae were dark, septate, about 3–8µm wide, very
dense, with clear edge and protruding from the
surface of the medium (Fig. 3A), being identified as
Phoma leveillei by using molecular techniques (Lü et
al. 2010), based on the result of the phylogenetic
analysis.
All of the six isolates of DSE were isolated from
the sample roots of cucumber plants in the last
fruiting stage of cucumber plants. Phoma leveillei
was the dominant species of DSE since it could be
isolated in all of the growth stages of cucumber
plants. F2‐2‐5 was appeared in the initial fruiting
stage, while the others were only isolated in the last
fruiting stage.
Different numbers of DSE isolates were found in
various cultivation region of cucumber. The isolates
F2‐1‐3, F2‐2‐5, and F2‐3‐10 of DSE were isolated in
Laiyang, F2‐1‐7, and F2‐2‐2 isolated in Shouguang,
and only one isolate F2‐2‐9 was found in Laixi.
Fig. 3 Two‐week‐old colony of the dark septate endophyte isolate on PDA. A: Front view; B: Reverse side.
Fig. 4 Neighbor‐joining phylogenetic tree based on ITS1‐5.8S‐ITS2 sequences of dark septate endophytes in the cucumber roots.
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The Kimura two‐parameter model was used for pairwise distance measurement. Numbers on branches were values generated
from 1 000 bootstrap replicates. Bootstrap values of 50% were shown above branch nodes.
3 DISCUSSION
AMF diversities have been extensively
investigated, and about 73%, 20%, and 7% of the
research reports on AMF community in roots, soil,
and coexistent in roots and soil respectively were
published from 2002 to 2013
(http://link.springer.com/). Quite intreseting DSE was
newly found commonly occurring on plant roots
colonized with AMF(Li & Guan 2007; Tian et al. 2015).
DSEs were present in all mycorrhizal and
non‐mycorrhizal plant species investigated
(Gucwa‐Przepióra et al. 2016). In the present
investigation we observed that both AMF and DSE
could colonise cucumber roots, even the same root
segment. Moreover, in the same site, AMF diversity
within roots of cucumber plants in various growth
stages was different. The species diversity of AMF and
DSE increased along with the development of
cucumber. This may be due to the influences of the
interactions between AMF or DSE colonization and
the development of plants in various growth stages
on root mycorrhizal colonization and extension. It
needs further study. On the other hand, how they
can infect the same roots and the interactions
between them are also in need of investigation.
The relative abundance of AMF in field soil and
roots determined by either morphological or
molecular approaches can be different. Molecular
analysis could not detect the presence of Glomus in
the case that the fungus was observed in roots
examined under the microscope (Shi et al. 2012).
Robinson‐Boyer et al. (2009) concluded that there
were limitations in use of nested PCR amplicons and
cloning approach for quantifying the relative
abundance of AMF in roots, but Shi et al. (2012)
proposed that there were several sources of
variability in quantification of AMF within roots
using both molecular and morphological methods. In
addition to the molecular detection, morphological
identification of spores isolated from the trap
culture using only the cucumber root segment as the
inoculum could also distinguish some AMF colonized
in the roots. Our results provide substantial evidence
of AMF community in root zone soil and within the
roots of cucumber plants grown in greenhouse soil,
and the results can be proved by molecular and
morphological identification. In our investigation of
the root samples from Laiyang, Laixi and Shouguang,
uncultured Archaeospora, Gigaspora margarita, and
G. fasciculatum were also examined, however,
distribution differences of the species require of
further research.
The diversity of AMF in root zone soil might be
greater than that in roots. We detected 20 species
from the soil samples, while only seven species
could be detected from the roots using molecular
identification, and just three species were isolated
from the trap culture inoculated with surface
sterilized cucumber root segments as inocula. Not
all species of AMF can colonise any plant roots
although AMF have chemotaxic abilities that enable
hyphal growth toward the roots of a potential host
plant (McGonigle & Miller 1999). For example, Cai et
al. (2008) isolated four AMF from the rhizospheric
soil and soil samples of Phellodendron amurense,
but only three AMF were detected from the root
samples by nested‐PCR. Experimental results
HU Yu‐Jin et al /Diversity of arbuscular mycorrhizal fungi and dark septate endophytes in the greenhouse cucumber roots and soil
菌物学报
11
indicate that there are some selectivities in the
symbiotic relationship between plants and AMF.
Further more, the growth and development stage of
plants, and the cultivation regions may also
influence the species distribution and diversities of
both AMF and DSE( see table 2, table3, and
paragraph 2.2). Thus, grasping at optimum
opportunity for isolation of more species of AMF or
DSE is important and this is in need of future study.
As in the other surveys on wild plants
(Reininger & Sieber 2012; Yan et al. 2014), we
observed and isolated DSE such as Phoma leveillei in
cucumber roots with or without colonization of AMF.
This indicates that the root of crops cultivated under
greenhouse conditions could also be colonised by
DSE. However, the number of DSE species isolated in
roots of plants grown in greenhouse might be
relatively less than that in wild plants. DSE diversity
in both plant roots and greenhouse soil, and the
specificity or the mutual selectivity between DSE
and plants need further study specially when both
AMF and DSE simultaneously colonise plant roots.
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（本文责编：韩丽）