Uploaded by John Ållen Beñas Dacuycuy

A comparison of fungal endophytic community diversity in tree leaves of rural and urban temperate forests of Kanto district, eastern Japan

advertisement
f u n g a l b i o l o g y 1 1 7 ( 2 0 1 3 ) 1 9 1 e2 0 1
journal homepage: www.elsevier.com/locate/funbio
A comparison of fungal endophytic community diversity in
tree leaves of rural and urban temperate forests of Kanto
district, eastern Japan
Emi MATSUMURA*, Kenji FUKUDA
Graduate School of Frontier Sciences, The University of Tokyo, 5-1-5 Kashiwa-no-ha, Kashiwa, Chiba 277-8563, Japan
article info
abstract
Article history:
To clarify the effects of forest fragmentation and a change in tree species composition fol-
Received 16 August 2012
lowing urbanization on endophytic fungal communities, we isolated fungal endophytes
Received in revised form
from the foliage of nine tree species in suburban (Kashiwa City, Chiba) and rural (Mt. Waga-
16 December 2012
kuni, Ibaraki; Mt. Takao, Tokyo) forests and compared the fungal communities between sites
Accepted 15 January 2013
and host tree species. Host specificity was evaluated using the index of host specificity (Si),
Available online 4 February 2013
and the number of isolated species, total isolation frequency, and the diversity index were
Corresponding Editor:
calculated. From just one to several host-specific species were recognized in all host tree spe-
Martin I. Bidartondo
cies at all sites. The total isolation frequency of all fungal species on Quercus myrsinaefolia,
Quercus serrata, and Chamaecyparis obtusa and the total isolation frequency of host-specific
Keywords:
species on Q. myrsinaefolia, Q. serrata, and Eurya japonica were significantly lower in Kashiwa
b diversity
than in the rural forests. The similarity indices (nonmetric multidimensional scaling (NMS)
Endophytic fungal community
and CMH) of endophytic communities among different tree species were higher in Kashiwa,
Forest isolation
as many tree species shared the same fungal species in the suburban forest. Endophytic
Host specificity
fungi with a broad host range were grouped into four clusters suggesting their preference
Urbanization
for conifer/broadleaves and evergreen/deciduous trees. Forest fragmentation and isolation
by urbanization have been shown to cause the decline of host-specific fungal species and
a decrease in b diversity of endophytic communities, i.e., endophytic communities associated with tree leaves in suburban forests were found to be depauperate.
Crown Copyright ª 2013 Published by Elsevier Ltd on behalf of The British Mycological Society. All
rights reserved.
Introduction
Forest types are largely determined by climate, namely temperature and precipitation (Whittaker 1970). It is well known
that the diversity of terrestrial plants is highest in the tropical
rain forests, whereas in temperate regions, a few tree species
dominate the forest canopy. Many fungi, such as decomposers, wood-decaying polypores, endophytes in the rhizosphere and phyllosphere, ectomycorrhizal (ECM) fungi, etc.,
depend upon forest plants. Fungal diversity per single host
in tropical forests is known to be higher than that in temperate forests; however, species diversity per forest is not
higher in the tropics for polypores (Drechsler-Santos et al.
dhiou et al. 2010), and arbuscular mycor2010), ECM fungi (Die
rhizal fungi (AMF) (Zhao et al. 2003). This is because most fungal groups show higher host specificity in temperate forests
than in tropical forests (Hattori 2005), and this is particularly
true for ECM fungi (Tedersoo & Nara 2010). Thus, ECM fungi
* Corresponding author. Tel./fax: þ81 4 7136 4750.
E-mail address: [email protected] (E. Matsumura).
1878-6146/$ e see front matter Crown Copyright ª 2013 Published by Elsevier Ltd on behalf of The British Mycological Society. All rights reserved.
http://dx.doi.org/10.1016/j.funbio.2013.01.007
192
display hyperdiversity in mixed conifer-broadleaf forest in
temperate regions, which is a consequence of host and fungal
diversity supporting each other (Ishida et al. 2007).
Endophytic fungi that asymptomatically infect healthy
plant tissues are known to be ubiquitous in every organ of every plant species (Petrini 1986). Most tree-leaf endophytes are
decomposers or weak pathogens, which latently infect the
leaves with horizontal infection by spores rather than systemic
infection (Wilson 1996; Kaneko & Kaneko 2004). Thus, abiotic
factors, such as temperature, rainfall, and humidity, and biotic
factors, such as host density, may strongly affect the occurrence of endophytic fungi (Carroll 1995; Schulz & Boyle 2005;
Sieber 2007). Under natural conditions, the tree-leaf endophyte
communities of tropical forests tend to be dominated by fungal
species that have a broad host range (Suryanarayanan et al.
2002; Schulz & Boyle 2005). In temperate forests, the endophytic communities in host species of the same plant family
tend to be dominated by closely-related endophyte species
(Petrini & Carroll 1981; Sieber et al. 1991; Sahashi et al. 2000;
Ragazzi et al. 2003; Helander et al. 2007; Hashizume et al.
2008). There are clear differences in endophytic fungal communities in temperate forests between gymnosperms and angiosperms, which are dominated by Helotiales and Diaporthales,
respectively. These two orders are estimated to have diverged
300 Ma ago, suggesting that the dominant endophytes have
coevolved with their hosts (Sieber 2007).
The number of species on an island is determined by a dynamic balance between immigration and extinction rates of species, which depend on the size of the island and the distance from
a source of propagules (MacArthur & Wilson 1967); this pattern is
true for endophytic fungi in birch leaves growing on islands
(Helander et al. 2007). This theory can also explain differences
in endophyte community characteristics between tropical and
temperate forests. Namely, in tropical forests, where tree species
are diverse and individuals of the same species are scattered,
host trees can be considered isolated islands for host-specific endophytic fungi, while the forest is a continuous mainland for
fungi with a broad host range. In contrast, in temperate and boreal forests that contain a few dominant tree species, hostspecific fungi can dominate the endophyte community.
Decreases in fungal abundance or species diversity in urban forests have been reported for many fungal groups
(Ochimaru & Fukuda 2007; Newbound et al. 2010; Bainard
et al. 2011), including leaf endophytes (Jumpponen & Jones
2010), but these reductions have only been attributed to direct
factors such as pollution and heat-islands. Habitat disjunction
and fragmentation, however, also influence not only the flora
and fauna (Iida & Nakashizuka 1995; Sewell & Catterall 1998),
but also the mycoflora (Drinnan 2005). We hypothesize that an
isolated environment will more strongly affect host-specific
fungal species according to the theory of island biogeography
(MacArthur & Wilson 1967). Specifically, endophytic fungi
with narrow host ranges will be more severely affected by limited dispersal than fungi with a broad host range.
Most temperate forests in Japan are mixed and codominated by tree species of diverse phylogeny (gymnosperms
and angiosperms) and different life forms (evergreen and deciduous). East Asian forests, including Japanese forests, are
more diverse than temperate forests in Europe and North
America. As a result, fungi with a broad host range might be
E. Matsumura, K. Fukuda
more frequently isolated in temperate forests in Japan than
in Europe and North America (Osono 2008; Hashizume et al.
2008). Therefore, Japanese temperate forests provide a good
model for examining the effect of fragmentation on both
host-specific fungi and species with a broad host range. Therefore, the aim of this study was to reveal the difference between
suburban and natural forests with respect to fungal endophytic
community diversity in temperate regions. We isolated leaf endophytic fungi from nine tree species including five common
species in three sites, a suburban forest and two rural forests.
Materials and methods
Materials and study sites
All tree leaves were sampled during the summer in 2006 and
2007 in two rural forests and a suburban forest (Mt. Wagakuni:
July 19, August 20, and September 21, 2007; Mt. Takao: August
20 and September 29, 2006, and July 26, 2007; Kashiwa: July 30
and September 7, 2006, and September 30, 2007; Table 1). In total, nine species from seven genera in four families, including
five tree species that were common to all sites, were surveyed
to determine endophytic communities (Table 1). All climatic
data were obtained from the nearest automated meteorological data-acquisition system (AMeDAS) weather station.
The Mt. Wagakuni (WG) site has a cool-temperate climate.
The upper part of the site was secondary forest dominated by
Carpinus spp., with some deciduous broadleaved trees. The
lower part comprises plantations of Japanese cedar [Cryptomeria japonica D. Don] and Hinoki cypress [Chamaecyparis obtusa
(Sieb. et Zucc.) Endl.], with Quercus myrsinaefolia Bl., Eurya
japonica Thunb., and Aucuba japonica Thunb. mixed in the
subtree layer. A climax stand of beech (Fagus crenata Blume)
was located next to the study site (Fig 1). Four Quercus species
(two evergreen species of subgenus Cyclobalanopsis, Q. myrsinaefolia and Quercus acuta Thunb., and two deciduous species
of subgenus Quercus (or Lepidobalanus), Quercus serrata and
Quercus crispula Bl.) occurred at this site.
The Mt. Takao (TK) site is characterized by both cool- and
warm-temperate climate. Evergreen conifers (Abies firma Sieb.
et Zucc. and Tsuga sieboldii Carr.) and evergreen broadleaved
Quercus spp. dominated the stands, either alone or in combination, with some stands of Fagus japonica Maxim. Plantations of
Japanese cedar and Hinoki cypress were located next to the study
area. In total, ten Quercus species (six evergreen species, Q. myrsinaefolia, Quercus glauca Thunb., Q. acuta, Quercus salicina Bl.,
Quercus sessilifolia Bl., Quercustakaoyamensis Makino, and three
deciduous species, Q. serrata, Q. crispula, and Quercusanguselepidola Nakai) occurred at this site (Yoshiyama 1985).
The Kashiwa (KS) site has a warm-temperate climate and is
an isolated 1.5-ha suburban forest (Fig 1). This stand is a secondary forest, dominated by evergreen and deciduous Quercus
spp. The succession of this forest will progress to climax evergreen Q. myrsinaefolia, Castanopsis sieboldii (Makino) Hatus. ex
T. Yamaz. et Mashiba, and Machilus japonica Sieb. et Zucc. ex
Bl. (e.g., Numata 1969). Stands were inferred to be approximately 60 y old (Yamashita et al. 2007). In this stand, some
planted conifer trees (C. japonica and C. obtusa) were also
found. In total, ten Quercus species (one evergreen,
Fungal endophytic diversity in urban forest
193
Table 1 e Site conditions and host tree species.
Site
Mt. Wagakuni
Land use
Location
Elevation (m asl)
Mt. Takao
Kashiwa
Rural
N36 190 1900
E140 120 0400
400
Rural
N35 370 3000
E139 140 3700
400
Urban
N35 540 0700
E139 560 0000
20
Climates (annual)
Average temperature ( C)
Max temperature ( C)
Min temperature ( C)
Rainfall (mm)
10.8
27
5.8
1330
12.7
28.9
3.4
1571.8
14.4
30.3
0.7
1335
Climates (sampling periods)
Average temperature ( C)
Max temperature ( C)
Min temperature ( C)
Average rainfall (mm)
22
32.5
14.3
178
22.4
33.7
15.8
240.3
25
34
17.5
180
Life form
Deciduous
Host species
Quercus serrata Murray
Castanea crenata Siebold et Zucc.
Fagus japonica Maxim.
Carpinus tschonoskii Maxim.
Mt. Wagakuni
þ
Mt. Takao
þ
Kashiwa
þ
þ
Evergreen
Eurya japonica Thunb. var. japonica
Quercus myrsinaefolia Blume
Quercus glauca Thunb.
þ
þ
þ
þ
þ
þ
þ
Conifer
Chamaecyparis obtusa (Siebold et Zucc.) Endl.
Cryptomeria japonica (L.f.) D.Don
þ
þ
þ
þ
þ
þ
Q. myrsinaefolia, and two deciduous trees, Q. serrata and Quercus acutissima Carruth.) occurred at this site.
We selected three individuals from each tree species, and
fifteen mature healthy leaves were collected from each selected individual during each sampling period. Therefore,
one sample unit comprised 135 leaf disks or segments (three
sampling periods 15 leaves three individuals from each
host species at each site).
Endophyte isolation
Fungal isolation was conducted within 48 h of sample collection. The leaves of collected samples were washed under running tap water for 12 h. Then, they were sterilized in 80 %
ethanol for 1 min, 1 % NaOCl for 1 min, 80 % ethanol for an additional 1 min, and then rinsed twice in sterile water. After
drying the leaves on sterile paper, two leaf disks (6 mm in diameter) were punched out from broadleaf species, Cryptomeria
needles were cut into 1-mm lengths, and a pair of Chamaecyparis scales were cut into 3-mm squares. The samples were incubated, with five segments per dish, on modified half-strength
potato dextrose agar (PDA) containing chloramphenicol (M-1/
2 PDA; 19.5 g PDA (Merck, Darmstadt, Germany), 9.0 g of plate
count agar (PCA; Merck), and 600 mg of chloramphenicol/L) in
a Petri dish (90-mm diameter) and kept at 20 C under dark
conditions. Emerged colonies were transferred into new Petri
dishes and incubated under the same conditions at room
temperature. The pure cultures were exposed to natural daylight if not sporulated. Sporulated isolates were identified
morphologically to genus or species level. Sterile isolates
were grouped into morphotypes according to their cultural
and morphological characteristics, and more than one isolate
from each morphotype was identified by DNA analysis.
þ
þ
Fungal identification by DNA analysis
Fungal DNA was extracted from pure culture mycelia using
a DNeasy Plant Mini Kit (Qiagen). The rDNA ITS region was
amplified using ITS5 and ITS4 primers (White et al. 1990).
PCR amplifications were performed in a reaction mixture containing AmpliTaq Gold master mix (Applied Biosystems) or
GoTaq master mix (Promega), DNA extracts, and the primer
pair. Thermocycler settings were 10 min initial denaturation
at 94 C, followed by 30 cycles of 30 s at 94 C, 1 min at 51 C,
1 min at 72 C, with a 10-min final extension at 72 C. The
PCR products were purified by Microcon-100 (Milipore) and
sequenced using a 3130 Genetic Analyzer (Applied Biosystems) after the sequence reaction with a BigDye Terminator
version 3.1 Cycle Sequencing kit. Aligned sequence data
were collected from BLAST searches. Isolates with over 97 %
sequence similarity were identified as the same species, and
those with over 95 % sequence similarity were identified as
the same genus. Sequence data were deposited in GenBank
(accession numbers are included in Table 5).
Data analysis
The isolation frequency (IF) of an endophyte taxon was calculated as follows: IF (%) ¼ Ni/Nt 100, where Ni is the total
number of leaf disks/segments from which the fungus was
isolated, and Nt is the total number of leaf disks/segments.
Fungal species with IF <3 % were regarded as rare. This included species of Cladosporium, Fusicoccum, Penicillium, Trichoderma, Biscogniauxia, Nodulisporium, Xylariaceous fungi,
yeasts, and unidentified fungi.
Host specificity was evaluated for each fungal taxon by the
index of host specificity (Si), which indicates both the number
194
E. Matsumura, K. Fukuda
Fig 1 e Study site, Satellite image of the Kanto area (left upper, Google Earth) and vegetation maps for each site (1 km square).
of host species and the evenness of their abundances as
(Rohde 1980)
X
X
Xij =nj ;
Si ¼
Xij =nj hij
where, for the ith fungal species, xij is the number of leaf segments where the ith species was isolated from the jth host species; nj is the number of leaf segments of the jth species
examined; and hij is the rank of host species j based on IF
(the species with the greatest IF has rank 1). After that, the distribution of the Si index was fitted to a mixture of normal distributions by JMP ver. 9.03.
The diversity of endophytic communities was evaluated by
use of the ShannoneWiener H0 and evenness index E5 (also
known as the modified Hill’s ratio). The E5 index was used
because it is relatively unaffected by the number of species
or the sample size (Ludwig & Reynolds 1988).
X pi loge pi
H0 ¼ l¼
X
p2i
0
E5 ¼ ð1=l 1Þ= eH 1 ;
where pi is the frequency of occurrence of each species.
To compare fungal diversity between sites, five tree species
that were common to all sites were used. To compare a diversity, the total number of fungal species in each site was
estimated from the species accumulation curve using ESTIMATES ver. 8.2 (Colwell 2006). To compare b diversity, we
Order
Botryosphaeriales
Xylariales
Diaporthales
Diaporthales
Helotiales
Capnodiales
Diaporthales
Pleosporales
Capnodiales
Diaporthales
Diaporthales
Diaporthales
Xylariales
Glomerellales
Diaporthales
Xylariales
Dothideales
Botryosphaeriales
Glomerellales
Pleosporales
Trichosphaeriales
Xylariales
Diaporthales
Mycosphaerellales
Glomerellales
Xylariales
Xylariales
Fungal species
Phyllosticta
cryptomeriae
White sterile Cs2
Gnomonopsis sp.
White sterile PH1
Tubakia sp. 2
Pezicula sp.
Pseudocercospora sp.
Tubakia sp. 1
Ascochyta fagi
Ramichloridium
cerophilum
Yellow sterile GL2
Discula sp. 1
Yellow sterile GL1
Tubakia sp. 3
Discula sp. 2
Rosellinia sp. 2
White sterile DT1
Glomerella sp.
(Colletotrichum sp. 3)
White sterile WM1
Phomopsis sp. 2
Xylaria sp.
Botryosphaeria dothidea
WHS
Phyllosticta capitalensis
Colletotrichum
acutatum
Alternaria sp.
Nigrospora sp. 2
Astrocystis sp.
Phomopsis spp.
White sterile WH7
Aureobasidium sp.
Colletotrichum
gloeosporioides
Muscodor
fengyangensis
Pestalotiopsis sp.
Cryptomeria
japonica
Cj
Si
WG
TK
KS
1.00
57.0
74.8
57.0
0.99
0.99
0.99
0.99
0.98
0.98
0.97
0.96
0.94
0.93
0.90
0.86
0.84
0.82
0.78
0.75
0.69
0.67
0.66
0.64
0.64
0.64
0.63
0.63
Chamaecyparis
obtusa
Co
WG
TK
KS
Eurya
japonica
Ej
WG
TK
Quercus
glauca
Qg
KS
TK
Quercus
myrsinaefolia
Qm
WG
TK
KS
7.8
þ
þ
7.4
11.9
þ
10.7
þ
14.8
33.0
12.2
19.3
1.5
13.0
7.0
6.7
þ
þ
þ
þ
þ
þ
43.7
þ
þ
þ
5.2
9.6
13.7
þ
þ
þ
11.1
11.1
4.4
þ
þ
þ
0.63
0.58
0.55
0.55
0.53
0.53
0.50
þ
5.9
6.7
5.9
þ
10.4
þ
0.48
33.3
0.43
þ
þ
9.6
14.1
þ
þ
þ
þ
4.4
þ
þ
9.6
þ
þ
25.2
þ
þ
5.2
4.4
5.2
þ
þ
þ
þ
TK
KS
WG
þ
þ
þ
11.5
48.1
þ
8.1
12.6
3.3
þ
þ
8.1
þ
10.4
6.7
þ
7.8
4.8
þ
þ
5.6
17.0
5.6
5.9
15.2
þ
þ
þ
þ
þ
17.0
þ
þ
5.2
52.2
þ
þ
15.6
47.4
5.6
5.6
þ
þ
þ
þ
4.8
þ
þ
þ
þ
7.8
þ
þ
þ
8.1
þ
þ
þ
þ
17.0
þ
þ
þ
þ
27.4
þ
þ
þ
þ
þ
þ
5.6
þ
þ
8.1
þ
þ
þ
þ
7.0
15.2
þ
þ
8.5
21.5
5.6
þ
þ
þ
þ
þ
þ
4.1
þ
8.1
5.9
21.1
5.6
7.8
þ
þ
þ
62.6
þ
þ
þ
28.9
þ
KS
þ
þ
þ
TK
þ
þ
þ
þ
WG
Fagus Castanea Carpinus
japonica crenata tschonoskii
Fj
Cc
Ct
þ
þ
þ
5.2
þ
þ
Quercus
serrata
Qs
58.5
þ
27.0
þ
þ
7.8
þ
þ
þ
þ
þ
14.8
þ
5.2
8.5
5.9
þ
þ
þ
43.0
14.4
þ
þ
þ
þ
4.8
þ
þ
31.1
6.3
þ
30.4
þ
þ
þ
þ
4.1
þ
þ
10.7
þ
þ
þ
54.8
þ
þ
þ
þ
þ
14.8
þ
17.4
þ
þ
þ
4.4
9.3
þ
4.8
þ
þ
þ
5.6
þ
11.5
þ
25.9
þ
þ
þ
þ
13.3
5.2
þ
þ
6.7
33.7
þ
þ
þ
9.6
3.3
4.4
þ
34.8
þ
þ
11.1
þ
þ
þ
þ
þ
þ
þ
4.8
7.0
þ
6.7
þ
7.8
23.7
þ
þ
7.0
þ
20.7
18.5
39.3
12.6
þ
3.7
þ
195
‘þ’ means IF lower than 3.0 %.
Host
species
Fungal endophytic diversity in urban forest
Table 2 e IF (%) and host-specificity index of major endophytic fungi on the leaves of nine tree species in three sites.
196
E. Matsumura, K. Fukuda
Table 3 e Comparison of the IF and diversity estimations for endophytes among three sites.
Host species site
Cryptomeria
japonica
WG
165
57.0
TK
110
74.8
KS
WG
TK
KS
Eurya japonica
WG
TK
Quercus
myrsinaefolia
KS
WG
102.2a 81.5ab 51.9b 110.0 98.5
110
7.4
14.8
9.6
13.3a 6.9ab 5.7b
TK
KS
Quercus serrata
WG
TK
KS
88.5ab 131.1a 75.9b 157.4ab 144.8a 136.7b
10.6b 30.4a 4.9b
62.6a
58.5a 27.0b
Sum of IF (%)
Sum of IFs of hostspecific species (%)
Sum of IFs of broadhost-range species (%)
Number of species
102.2a 21.5b 37.8ab 85.2a
51.1ab 33.3b 58.9
69.3
88.1
50.7a
33.0b
55.2a 78.1
76.3
95.9
17
27
24
24
31
23
31
27
30
Chao2
Jaccard2
Completedness in %
24
25
17
28
50
29
32
18
35
52
59e71 56e72 89e96 60e75 52e55
22
68
26
26
69
45
54
69
27
54
31
29
64
42
56
64
67e83 43e54 77e91 83e94 45e49 51e55 56e58 45e49
38
35
44
41
61e71 73e85
Diversity indices (H0 )
Evenness (E5)
1.78
0.65
2.53
0.77
2.16
0.48
18
1.38
0.36
104
57.0
Chamaecyparis
obtusa
16
1.76
0.43
21
2.19
0.60
18
2.75
0.70
29
2.63
0.73
2.46
0.68
2.33
0.64
2.38
0.61
2.00
0.57
2.62
0.63
31
2.15
0.46
2.54
0.56
Letters indicate significant differences among sites (SteeleDwass test, a ¼ 0.05).
calculated the similarity of fungal composition among the different host species at each site. The MorisitaeHorn similarity
index (CMH) was used to assess similarity in ESTIMATES.
X
ðani bni Þ=ðda þ dbÞaN bN;
CMH ¼ 2
where aN is the total number of isolates in host species A, ani
is the number of isolates of the ith species in host species A, bni
is the number of isolates of the ith species in host species B,
P
P
da ¼ an2i =aN2 and da ¼ bn2i =aN2 .
A Bonferroni correction was applied to the paired nonmetric variance (Wilcoxon signed-rank test) for multiple comparisons of CMH between sites. Nonmetric comparisons by the
Table 4 e Dominant fungal endophytes in this study and previous studies (for host code, see Table 3).
Present study
Previous studies
Host species
Dominant species
Order
Host species
Qs*
Discula sp.
Phomopsis spp.
Diaporthales
Diaporthales
Qs
Discula sp.
Phomopsis sp.
Diaporthales
Diaporthales
Matsuda et al. 2010
Qm*, Qg
Tubakia sp. 1
Tubakia sp. 2 (QA-b)
Tubakia sp. 3
Diaporthales
Diaporthales
Diaporthales
Quercus acuta
QA-b (Tubakia sp.)
Discula sp.
Phomopsis sp.
Diaporthales
Diaporthales
Diaporthales
Hashizume et al. 2008
Ej*
C. gloeosporioides
Phy. capitalensis
R. cerophilum
Glomerellales
Botryosphaeriales
Capnodiales
Camellia japonica
C. gloeosporioides
Geniculosporium sp. 1
C. acutatum
Glomerellales
Xylariales
Glomerellales
Osono 2008
Co*
Xylaria sp.
Xylariales
Chamaecyparis
lawsoniana
Scolecosporiella sp.
Pleosporales
Petrini & Carroll 1981
Pezicula sp.
Helotiales
Nodulisporium sp.
Geniculosporium sp.
Chloroscypha alutipes
Pezicula sp.
Xylariales
Xylariales
Helotiales
Helotiales
Cj*
Phy. cryptomeriae
Xylaria sp.
Botryosphaeriales
Xylariales
Fj
Glomerella sp.
Phomopsis spp.
C. gloeosporioides
Glomerellales
Diaporthales
Glomerellales
Discula sp.
Ascochyta fagi
Diaporthales
Pleosporales
Cc
Phy. capitalensis
Phomopsis spp.
C. gloeosporioides
Botryosphaeriales
Diaporthales
Glomerellales
Ct
Gnomonopsis sp.
Phomopsis spp.
Diaporthales
Diaporthales
Fagus crenata
Order
References
Sahashi et al. 2000
Hashizume et al. 2010
Alnus rubra
Betula pubescens,
B. pendula
*Host common to all sites.
Dominant species
Gnomonia setacea
Gnomoniella tubiformis
Fusicladium betulae
Gnomonia setacea
Melanconium betulinum
Diaporthales
Diaporthales
Pleosporales
Diaporthales
Diaporthales
Sieber et al. 1991
Helander et al. 2007
Fungal endophytic diversity in urban forest
197
Table 5 e BLAST search results for major endophytic fungi.
Fungal species
Phyllosticta cryptomeriae
White sterile Cs2
Gonomonopsis sp.
White sterile PH1
Tubakia sp. 2
Pezicula sp.
Pseudocercospora sp.
Tubakia sp. 1
Ascochyta fagi
Ramichloridium cerophilum
Yellow sterile GL2
Discula sp. 1
Yellow sterile GL1
Tubakia sp. 3
Discula sp. 2
Rosellinia sp. 2
White sterile DT1
Glomerella sp. (Colletotrichum sp. 3)
White sterile WM1
Phomopsis sp. 2
Xylaria sp.
Botryosphaeria dothidea
WHS
Phyllosticta capitalensis
Colletotrichum acutatum
Alternaria sp.
Nigrospora sp. 2
Astrocystis sp.
Phomopsis spp.
White sterile WH7
Aureobasidium sp.
Colletotrichum gloeosporioides
Muscodor fengyangensis
Pestalotiopsis sp.
Accession number
BLAST search result
Accession number
Score (%)
AB731136
Phyllosticta cryptomeriae
AB454271
629/632 (99 %)
AB731135
Gnomonopsis sp.
JN793536
539/564 (96 %)
AB731134
AB731133
Dicarpella dryina
Pezicula sp.
JF502454
AF141173
531/591 (90 %)
517/531 (97 %)
AB731132
QA-b
AB365875
522/600 (87 %)
AB731131
Ramichloridium cerophilum
HQ608156
473/480 (99 %)
AB731130
Rosellinia sp.
HQ907947
513/519 (99 %)
AB731129
AB731128
Glomerella lindemuthiana
Fungal endophyte sp.
FN566869
AB255246
571/573 (99 %)
510/512 (99 %)
AB731127
AB731126
Xylaria sp.
Botryosphaeria dothidea
HM595549
AB645751
555/555 (100 %)
557/558 (99 %)
AB731124
AB731123
Guignardia mangiferae
Colletotrichum acutatum
AB454332
AJ301905
628/628 (100 %)
542/550 (99 %)
AB731121
Astrocystis cocoes
AY862571
457/478 (96 %)
AB731122
Muscodor fengyangensis
HM034852
551/564 (98 %)
SteeleDwass test were used to detect differences in IF and the
number of isolates of fungi with a broad host range.
Relationships between host-specific groups, broad-hostrange groups, and whole fungi were evaluated using the nonparametric Spearman’s correlation test. JMP ver. 9. 03 was
used for these statistical analyses.
Multiple classification analysis and nonmetric multidimensional scaling (NMS) were performed for 57 fungal communities (units) in the 57 trees using the Sørensen index and
50 randomized runs.
Fungal taxa that had a broad host range were grouped
according to their host preference through two-way cluster
analysis, with their relative dominance based on the IF of
each host species in each site. PC-ORD ver. 5 was used for
the community analysis.
Results
3 % in each sample unit (i.e., host tree species in each site). The
host-specificity index (Si) for the 34 frequent morphotypes
ranged from 0.43 to 1.00. It consisted of a mixture of four normal distributions. Thus, a value of 0.72, which divided the second and third distributions, was used as the border between
host-specific species and species with a broad host range.
Overall, seven species in 11 genera with Si values higher
than 0.72 were defined as host-specific fungi (Table 2).
All tree species had at least one host-specific endophyte
species, which were isolated from the specific host species
at all sites. Host-specific species were Phyllosticta cryptomeriae
and Rosellinia sp. 2 in Cj (for host code, see Table 2), Pezicula
sp. and White DT1 in Co, Ramichloridium cerophilum and Yellow
GL1e2 in Ej, PH1 in Qg, Tubakia sp. 3 (‘QA-b’ in Hashizume et al.
2008) in Qm and Qg, Tubakia sp. 1e2 in Qm, Discula sp. 1 in Qs
and Fj, Discula sp. 2 in Cc, Ascochyta fagi in Fj, and Gnomonopsis
sp., Cs2, and Pseudocercospora sp. in Ct. In Fagaceae, six hostspecific species belonged to Diaporthales.
Frequency of fungal species and their host specificity
IF, fungal richness, and diversity
In total, 6071 isolates were detected from 4320 leaf segments
taken from the three sites. The isolates were classified into
128 morphotypes. Thirty-four morphotypes (fungal species;
Table 5) were distinguished as major taxa, and the other 94
morphotypes were defined as rare fungi, whose IF was under
The sum of the IFs for all fungi in the hosts Co, Qm, and Qs was
significantly lower in the suburban forest than in the two rural
forests. The other two host species exhibited the same trend, although the difference was not significant (Table 3). The sum of
the IFs of host-specific species in Ej, Qm, and Qs was significantly
198
E. Matsumura, K. Fukuda
lower in the suburban forest than in the rural forests, and the
other two host species also exhibited the same trend (Table 3).
Moreover, the sum of the IFs of species with a broad host range
in Cj, Co, and Qm was also significantly higher in the rural WG
site and was lowest in the suburban forest.
In Cj and Co, the sum of the IFs of species with a broad host
range exhibited strong positive correlations with the sum of
the IFs of all fungal species (Cj, 0.72, Co, 0.78; Spearman’s correlation coefficient, P < 0.0001). On the other hand, in Co, Ej, Qm,
and Qs, the sum of the IFs of species with a broad host range
exhibited negative correlations with the sum of the IFs of hostspecific species (Co, 0.45, Ej, 0.63, Qm, 0.45, Qs, 0.62;
P < 0.02). Although the number of fungal species and fungal diversity (H0 ) did not differ significantly among sites for each host
species, species accumulation (rarefaction) curves indicated
that fungal richness and the number of isolations were lowest
in the suburban forest (Fig 5).
variation, and the fungal communities were separated according to host species and site (Fig 2; Stress ¼ 14.1, P < 0.02). Fungal communities on the same host displayed less variation;
however, fungal communities on different hosts at the same
site were sometimes less varied than those on allopatric conspecific hosts (e.g., Qm and Ej at KS and WG; Cj and Co at WG).
Fungal communities were more strongly influenced by sympatry on some hosts or in some sites.
To assess the similarity of fungal communities among tree
species within a site, we calculated the volume of a polyhedron formed by five points consisting of five tree species common to all three sites. The volumes were 0.58 at WG, 0.70 at
TK, and 0.36 at KS (Fig 2), which suggested that the fungal
communities among tree species in the suburban forest
were most similar. This result was confirmed by the CMH
among sites, which was significantly higher for KS than for
TK (paired nonparametric Wilcoxon signed-rank test,
a < 0.05, Bonferroni correction).
Fungal communities
Fungi with a broad host range
To summarize and visualize the compositional differences in
fungal communities among host species and sites, we used
NMS. Three axes accounted for a substantial 78.5 % of the
Chamaecyparis obtuse
A1
C
C. acutatum
C. gloeosporioides
Quercus myrsinaefolia
3
C. sp.3
Eurya japonica
4
Phy. capitalensis
Quercus serrata
5
Phomopsis spp.
6
Phomopsis sp.2
7
WM1
8
Pestalotiopsis sp.
9
Nigrospora sp.2
Quercus glauca
Carpinus tschonoskii
0
1
2
Fagus japonica
Axis 3 (42.5%)
1.5
Cryptomeria japonica
Castanea crenata
Mt. Takao
Mt. Wagakuni
Kashiwa
Qm
Qm
Axis 3 (42.5%)
1.5
Fungal taxa with a Si lower than 0.72 were defined as species
with a broad host range. They were not always isolated from
Fj
Ej
Ej
Qm
4 1
3
2
Cc
0
12
7
6
5
Qs
10 A. cocoes
8
11
910
13
Ct
Ct
11 WH7
Co
12 M. fengyangensis
13 Xylaria sp.
Co
Cj
Cj
-1.5
-1.5
0
-1.5
Axis 2 (24.1%)
1.5
1.5
Axis 2 (24.1%)
1.5
B1
0
B2
Axis 3 (42.5%)
A2
Axis 3 (42.5%)
0
-1.5
1.5
D
Qm
Qm
Ct
Ct
Fj
14
Qm
6
5
0
Ej
Ej
3
2
Cc
7
Qs
12 11
10
8
9
Co
Co
13
Cj
Cj
-1.5
-1.5
0
Axis 1 (11.8%)
1.5
-1.5
-1.5
0
1.5
Axis 1 (11.8%)
Fig 2 e Nonmetric multidimensional scaling (NMS) of fungal species isolated in nine tree species at three sites. Each figure
represents a sample unit (A, B) or fungal species (C, D). The letter indicates the specific host species in C, D. The similarity of
fungal communities is compared for three sites (A-1, B-1).
Fungal endophytic diversity in urban forest
100
B
C
D
Similarity (%)
50
75
100
25
0
80
Phomopsis spp.
Phomopsis sp.2
Alternaria sp.
WM1
Astrocystis sp.
WHS
C. gloeosporioides
C. acutatum
P. capitalensis
Glomerella sp.
Xylaria sp.
M. fengyangensis
Pestalotiopsis sp.
Nigrospora sp.2
WH7
B. dothidea
Aureobasidium sp.2
Number of species
A
199
60
40
WG
20
TK
KS
0
Fig 3 e Isolation trend for broad-host-range fungi by cluster
analysis.
0
200
400
600
800
1000
Number of isolates
Isolation frequency (%)
Fig 5 e Rarefaction curves of the endophytic fungi in three
sites for five common host species. WG: Mt. Wagakuni; TK:
Mt. Takao; KS: Kashiwa.
150
a
a
100
b
b
50
a
a
b
b
b
C
D
A
b
b
b
0
A
B
Deciduous
B
C
D
A
Evergreen
B
C
D
Conifer
Mt. Wagakuni
150
a
100
b
50
a
b
b
c
b
c
0
A
B
C
D
Deciduous
A
B
C
D
A
Evergreen
B
C
all sites. The dendrogram resulting from the two-way cluster
analysis (Fig 3) at 30 % similarity produced four main clusters,
which were characterized by the following fungal groups:
cluster A, Phomopsis spp., frequently isolated from deciduous
broadleaves; cluster B, Colletotrichum spp. from evergreen
broadleaves; cluster C, Xylariaceae; and cluster D, Pestalotiopsis
sp., from conifers in WG and KS. These four groups of fungi
with a broad host range are shown in Fig 4, and their distribution varied according to the host-species life forms. This trend
was consistent for NMS fungal plots (Fig 2C and D). We compared the number of isolates in the four clusters among sites.
At WG, where conifers and evergreens were sympatric, cluster
C fungi were more frequently found in evergreen hosts. At TK,
which was dominated by evergreen trees, fungi belonging to
cluster B showed a higher frequency in deciduous hosts
than at other sites. At KS, a mixed evergreen and deciduous
forest, cluster A in evergreen and cluster B in deciduous hosts
were more frequent than in other sites. Cluster D in deciduous
hosts was more frequent than cluster C at KS.
D
Discussion
Conifer
Mt. Takao
Host specificity
150
100
a
a
a
50
b
b
c
c
b
D
A
c
ab ab
a
B
D
0
A
B
C
D
Deciduous
A
B
C
Evergreen
C
Conifer
Kashiwa
Fig 4 e Boxewhisker plots comparing ranked medians of IF
of fungal groups with a broad host range in nine tree species at three sites. Cluster A, grey; cluster B, open; cluster C,
dark grey; cluster D, slashes, see Fig 3. Different letters indicate significant differences among the four fungal groups
(SteeleDwass test, a [ 0.05).
To identify differences in fungal endophyte communities between suburban and natural forests, we investigated the fungal diversity of suburban and rural forest sites in Japan and
considered the endophytic communities in conspecific tree
leaves. All host species studied had one to three hostspecific species, which often dominated in their host. When
compared to previous studies of the same or closely-related
hosts worldwide, many common or similar fungal taxa were
shown to be dominant regardless of geographical differences
(Table 4). Our study is the first detailed report of endophytic
fungal communities and their host tree species, with the exception of Quercus serrata. For Q. serrata, the dominant fungi
(Discula sp. and Phomopsis sp.) were the same as those identified by Matsuda et al. (2010) in western Japan. Endophytic
communities in Fagaceous and Betulaceous trees were dominated by Diaporthales. Quercus spp. in particular exhibited
200
a high affinity with Diaporthales. On the other hand, communities in Fagaceae, except for the Quercus genus, were frequently
dominated by Colletotrichum spp. and Phyllosticta capitalensis,
which are recognized as common fungi of evergreen hosts.
This may suggest that forest type also affects endophytic
dominance.
In tropical forests, Suryanarayanan (2011) showed that the
infection frequency of Phomopsis spp. gradually decreased
when forests became wetter and the infection frequency of
Colletotrichum spp. increased with increasing annual rainfall
before reaching its maximum in an evergreen forest that received the greatest rainfall of the forests studied. In our study,
Colletotrichum spp. and Phy. capitalensis were more frequently
isolated from evergreen trees, and Phomopsis spp. were more
frequently isolated from broadleaved trees (Fig 4). Our results
are consistent with Suryanarayanan (2011) in terms of the
combinations of host life forms and fungal genera. This might
suggest that their abundance could be influenced by the
life form of the dominant hosts (deciduous or evergreen)
rather than rainfall or humidity. Differences in rainfall and
humidity among our study sites were almost negligible compared to Suryanarayanan (2011). Additionally, Xylariales were
frequently isolated from two conifer species (Fig 4), which
supports the findings of a previous study of Cupressaceae
€ ller 1979; Petrini & Carroll 1981; Bills &
(Petrini & Mu
Polishook 1992; Hoffman & Arnold 2008). Therefore, we conclude that these fungi with a broad host range also have
some host preferences.
Decrease in endophyte diversity in suburban forest (KS)
In the suburban forest, the IF values for endophytes as a whole
and for host-specific fungi were both lower than in rural forests. The decrease in host-specific fungi and the increase in
fungi with a broad host range resulted in the highest CMH being observed between tree species in the suburban forest,
namely, a low b diversity of endophytic communities in suburban forest. In the same Kanto area, Ochimaru & Fukuda
(2007) compared a mushroom community across a gradient
from urban to rural areas and demonstrated that species richness and the frequency of ECM fungi were negatively correlated with urbanization. Urbanization can cause not only
microclimate changes, including heat-island effects, and
chemical changes because of pollution, but also forest fragmentation and isolation. However, previous reports have not
referred to forest isolation.
Helander et al. (2007) studied leaf endophytes in Betula spp.
in an archipelago in Finland and showed that total isolation frequencies of endophytes decreased with increasing distance
from the mainland and decreasing size of the island. Our study
was consistent with this result if we regard the fragmented forests in a suburban area as islands in a city. Furthermore,
Drinnan (2005) clarified fragmentation thresholds by urbanization for multibiota with different trophic levels. The study indicated a potential threshold in a reserve of approximately 2 ha in
size, below which plant and fungal species richness decreased
rapidly. The suburban forest in our study was approximately
1.5 ha in size and was also isolated from other forests (Fig 1). Although fungal species richness and the diversity index per single host species (a diversity) among the three sites did not
E. Matsumura, K. Fukuda
differ, the lower total IF and lower b diversity in KS could be attributed to limited spore dispersal in fragmented forests.
Positive relationships between host density and the frequency of dominant endophytic fungi, especially in host€ ller & Hallaksela
specific species, have been reported (Mu
1998; Helander et al. 2006). In our study, Quercus serrata and
Quercus myrsinaefolia were dominant in the suburban forest;
however, the frequency of their dominant endophytes, Discula
sp. and Tubakia spp., was lower than in the rural forests. This
may be because the number of tree species belonging to the
genus Quercus was lowest in the suburban forest (one evergreen species and two deciduous species at KS; two and two
at WG; seven and three at TK), and the total dominance of
these oak species (i.e., an infection source) in the suburban
forest was the lowest of the three sites. This trend was also observed in other host species.
There are many possible explanations for the difference in
endophytic communities between suburban and rural forests.
Both urbanization and forest fragmentation can lead to decreases in the diversity of endophytic communities. The experimental design used in this study does not allow us to
distinguish the effects of fragmentation from other factors,
such as microclimatic changes or pollution; however, these
various influences of urbanization on host-specific fungi and
fungi with a broad host range were observed not only in this
study but also in other urban forests (E.M. & K.F., unpubl.
data). These declines in host-specific fungi in urban forests
support our hypothesis that forest fragmentation and isolation in urban areas affect the endophytic community.
In conclusion, the endophytic community in temperate
forests is characterized by high host specificity, namely high
b diversity among host species. The infection rate of fungi
with a broad host range also exhibits some host preference.
However, in isolated suburban forests, the number of hostspecific endophytes decreases, and the frequency of fungi
with a broad host range increases. Endophytic communities
in suburban forest have been shown to be depauperate, with
low b diversity. This may be a result of the decreased infection
source of host-specific fungi in isolated urban forests.
Acknowledgement
The authors thank Dr Y. Hashizume for help with the sampling design and sampling. We also thank Dr Y. Yaguchi for
fungal identification and Dr N. Sahashi, Dr T. Umebayashi
and Dr A. Oda-Tanaka for their advice and support.
Appendix A. Supplementary data
Supplementary data related to this article can be found at
http://dx.doi.org/10.1016/j.funbio.2013.01.007.
references
Bainard LD, Klironomos JN, Gordon AM, 2011. The mycorrhizal
status and colonization of 26 tree species growing in urban
and rural environments. Mycorrhiza 21: 91e96.
Fungal endophytic diversity in urban forest
Bills GF, Polishook J, 1992. Recovery of endophytic fungi from
Chamaecyparis thyoides. Sydowia 44: 1e12.
Carroll G, 1995. Forest endophytes e pattern and process. Canadian Journal of Botany 73: S1316eS1324.
Colwell RK, 2006. EstimateS: statistical estimation of species richness
and shared species from samples Version 8. User’s Guide and
Application published at: http://purl.oclc.org/estimates (accessed 04.09.11)
dhiou AG, Selosse MA, Galiana A, Diabate
M, Dreyfus B, Ba
^ AM,
Die
na G, 2010. Multi-host ectomycorrhizal fungi
De Faria SM, Be
are predominant in a Guinean tropical rainforest and shared
between canopy trees and seedlings. Environmental Microbiology 12: 2219e2232.
Drechsler-Santos ER, Santos PJP, Gibertoni TB, Cavalcanti MAQ,
2010. Ecological aspects of Hymenochaetaceae in an area of
Caatinga (semi-arid) in northeast Brazil. Fungal Diversity 42:
71e78.
Drinnan IN, 2005. The search for fragmentation thresholds in
a southern Sydney suburb. Biological Conservation 124: 339e349.
Hashizume Y, Fukuda K, Sahashi N, 2010. Effects of summer
temperature on fungal endophyte assemblages in Japanese
beech (Fagus crenata) leaves in pure beech stands. Botany 88:
266e274.
Hashizume Y, Sahashi N, Fukuda K, 2008. The influence of altitude on endophytic mycobiota in Quercus acuta leaves collected in two areas 1000 km apart. Forest Pathology 38: 218e226.
Hattori T, 2005. Diversity of wood-inhabiting polypores in temperate forests with different vegetation types in Japan. Fungal
Diversity 18: 73e88.
Helander ML, Ahlholm JU, Sieber T, Hinneri S, Saikkonen K, 2007.
Fragmented environment affects birch leaf endophytes. New
Phytologist 175: 547e553.
€ li P, Kuuluvainen T, Saikkonen K, 2006. Birch leaf
Helander ML, Wa
endophytes in managed and natural boreal forests. Canadian
Journal of Forest Research 36: 3239e3245.
Hoffman MT, Arnold AE, 2008. Geographic locality and host
identity shape fungal endophyte communities in cupressaceous trees. Mycological Research 112: 331e344.
Iida S, Nakashizuka T, 1995. Forest fragmentation and its effect
on species diversity in sub-urban coppice forests in Japan.
Forest Ecology and Management 73: 197e210.
Ishida T, Nara K, Hogetsu T, 2007. Host effects on ectomycorrhizal
fungal communities: insight from eight host species in mixed
coniferebroadleaf forests. New Phytologist 174: 430e440.
Jumpponen A, Jones KL, 2010. Seasonally dynamic fungal communities in the Quercus macrocarpa phyllosphere differ between urban and nonurban environments. New Phytologist
186: 496e513.
Kaneko R, Kaneko S, 2004. The effect of bagging branches on
levels of endophytic fungal infection in Japanese beech leaves.
Forest Pathology 34: 65e78.
Ludwig JA, Reynolds JF, 1988. Statistical Ecology. A Primer on
Methods and Computing. John Wiley and Sons, NY, USA, pp. 337.
MacArthur RH, Wilson EO, 1967. The Theory of Island Biogeography.
Princeton University Press, NJ, USA, pp. 203.
Matsuda Y, Ito Y, Ito S, 2010. Infection patterns of endophytic
fungi in different leafing stages of Quercus serrata. Tree and
Forest Health 14: 165e173 (in Japanese with English summary).
€ ller MM, Hallaksela A, 1998. Diversity of Norway spruce needle
Mu
endophytes in various mixed and pure Norway spruce stands.
Mycological Research 102: 1183e1189.
Newbound M, McCarthy MA, Lebel T, 2010. Fungi and the urban
environment: a review. Landscape and Urban Planning 96: 138e145.
Numata M, 1969. Progressive and retrogressive gradient of
grassland vegetation measured by degree of succession. Vegetatio 19: 96e127.
201
Ochimaru T, Fukuda K, 2007. Changes in fungal communities in
evergreen broad-leaved forests across a gradient of urban to
rural areas in Japan. Canadian Journal of Forest Research 37:
247e258.
Osono T, 2008. Endophytic and epiphytic phyllosphere fungi of
Camellia japonica: seasonal and leaf age-dependent variations.
Mycologia 100: 387e391.
Petrini O, Carroll G, 1981. Endophytic fungi in foliage of some
Cupressaceae in Oregon. Canadian Journal of Botany 59:
629e636.
€ ller E, 1979. Pilzliche Endophyten, am Beispiel von
Petrini O, Mu
Juniperus communis L. Sydowia 32: 224e225.
Petrini O, 1986. Taxonomy of endophytic fungi of aerial plant
tissues. In: Fokkema NJ, Van den Heuvel J (eds), Microbiology of
the Phyllosphere. Cambridge University Press, Cambridge, UK,
pp. 175e187.
Ragazzi A, Moricca S, Capretti P, Dellavalle I, Turco E, 2003. Differences in composition of endophytic mycobiota in twigs and
leaves of healthy and declining Quercus species in Italy. Forest
Pathology 33: 31e38.
Rohde K, 1980. Host specificity indices of parasites and their application. Experientia 36: 1369e1371.
Sahashi N, Miyasawa Y, Kubono T, Ito S, 2000. Colonization of
beech leaves by two endophytic fungi in northern Japan. Forest
Pathology 30: 77e86.
Schulz B, Boyle C, 2005. The endophytic continuum. Mycological
Research 109: 661e686.
Sieber TN, Sieber-Canavesi F, Dorworth CE, 1991. Endophytic
fungi of red alder (Alnus rubra) leaves and twigs in British
Columbia. Canadian Journal of Botany 69: 407e411.
Sieber T, 2007. Endophytic fungi in forest trees: are they mutualists? Fungal Biology Reviews 21: 75e89.
Suryanarayanan TS, 2011. Diversity of fungal endophytes in
€ AM, Frank AC (eds), Endophytes of
tropical trees. In: Pirttila
Forest Trees Biology and Applications. Springer, New York,
pp. 67e80.
Suryanarayanan TS, Murali T, Venkatesan G, 2002. Occurrence
and distribution of fungal endophytes in tropical forests
across a rainfall gradient. Canadian Journal of Botany 80:
818e826.
Sewell SR, Catterall CP, 1998. Bushland modification and styles of
urban development: their effects on birds in south-east
Queensland. Wildlife Research 25: 41e63.
Tedersoo L, Nara K, 2010. General latitudinal gradient of biodiversity is reversed in ectomycorrhizal fungi. New Phytologist
185: 351e354.
White TJ, Bruns T, Lee S, Taylor JW, 1990. Amplification and direct
sequencing of fungal ribosomal RNA genes for phylogenetics.
In: Innis MA, Gelfand DH, Sninsky JJ, White TJ (eds), PCR Protocols: a guide to methods and applications. Academic Press Inc.,
New York, pp. 315e322.
Whittaker RH, 1970. Communities and Ecosystems. Macmillan,
London, 162 pp.
Wilson D, 1996. Manipulation of infection levels of horizontally
transmitted fungal endophytes in the field. Mycological Research 100: 827e830.
Yamashita S, Fukuda K, Ugawa S, 2007. Ectomycorrhizal communities on tree roots and in soil propagule banks along
a secondary successional vegetation gradient. Forest Science 53:
635e644.
Yoshiyama H, 1985. Habitat segregation between evergreen oak
and beech on Mt. Takao. Tama no Shizen 82: 18e23 (in
Japanese).
Zhao Z, Wang G, Yang L, 2003. Biodiversity of arbuscular mycorrhizal fungi in a tropical rainforest of Xishuangbanna, southwest China. Fungal Diversity 13: 233e242.
Download
Random flashcards
State Flags

50 Cards Education

Countries of Europe

44 Cards Education

Art History

20 Cards StudyJedi

Sign language alphabet

26 Cards StudyJedi

Create flashcards