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Received: 24 October 2015    Accepted: 22 September 2016

DOI: 10.1111/efp.12322

ORIGINAL ARTICLE

Assessment of the pathogenicity of marine Cladosporium spp.

towards mangroves

Y. Liu | Y. Li | Q. Lin | Y. Zhang

Guangdong Ocean University, Zhanjiang,

Guangdong, China
Correspondence
Yuelian Liu, Guangdong Ocean University,
Zhanjiang, Guangdong, China.
Email: mushwoman@126.com
Editor: J. Stenlid
Funding information
Natural Science Fund of Guangdong,
Grant/Award Number: 2016A030313746
and Doctoral Fund of Guangdong Ocean
University, Grant/Award Number: E15037
Summary
Mangroves play an important role in coastal ecosystems. In this work, Cladosporium
spp were isolated from a natural mangrove environment in Zhanjiang Bay, Guangdong
Province, China. Ninety-­one isolates were obtained and identified as Cladosporium cla-
dosporioides, C. colocasiae, C. oxysporum, C. perangustum, C. sphaerospermum, C. tenuis-
simum and C. uredinicola. Forty-­two isolates were selected and tested for pathogenicity
towards mangroves. The results showed that pathogenic isolates were found on
Avicennia marina, Kandelia candel and Rhizophora stylosa with prevalence of 93.40%,
83.52% and 7.41%, respectively. This indicated that A. marina and K. candel were more
vulnerable to Cladosporium ssp. than R. stylosa. Among the seven species, C. colocasiae
had the strongest pathogenic effect towards A. marina and K. candel with a prevalence
of 100.00%. The rest of the seven species also showed pathogenic effects on the same
plants. These results provided valuable insights into the effect of various pathogens on
mangroves.

1 |  INTRODUCTION Cladosporium cladosporioides (Fresenius) de Vries was also isolated

Cladosporium sp. is a common terrestrial plant pathogen (Curtis,
Gore, & Oliver, 1994; Schubert, Braun, Groenewald, & Crous, 2007).
Recently, several studies have isolated Cladosporium sp. from marine
environments (Butinar, Sonjak, Zalar, Plemenitaš, & Gunde-­Cimerman,
2005; Devi, Rajkumar, & Beenish, 2014; Fuad, Mohamed, Sarfaraz, &
Hadi, 2015; Zalar et al., 2007; Zhang, Zhang, Xu, & Qi, 2013). Also,
in our study, Cladosporium sp. was identified as the most dominant
genus in mangroves in Zhanjiang Bay, Guangdong Province of China
(Liu, Zhang, Hu, & Wang, 2013; Liu, Zhang, Lin, Hu, & Wang, 2015). It
must be noted that the mangrove ecosystem has high genetic diversity
due to the differences between aquatic and terrestrial microbial com-
munities (Cao, Zheng, Chen, & Chen, 2008).
According to literature, mangroves are known to be affected by
various terrestrial contaminations (Aziz, Khairul, Chew, Ving, & Kwai,
2014; Joshi & Ghose, 2014; Peng et al., 2005; Touron et al., 2007).
Additionally, it was reported that some common terrestrial pathogens
such as Cytospora rhizophorae (Wier & Tarrar, 2000); Colletotrichum
sp., Alternaria sp. and Phyllosticta sp. (Zhou & Huang, 2001); and
Pestalotiopsis versicolor (Zhang et al., 2009) also infected mangroves.
Another report showed that the filamentous fungus identified as
from marine alga in vitro (Cooley, Mullins, Bradley, & Wilce, 2011).
The mangrove ecosystem is widely distributed in tropical and sub-
tropical seacoasts, playing an important role in withstanding storms,
pollution remediation and protection of marine organisms (Gopal &
Chauhan, 2006; Kathiresan, 2000; Lin, 1997). However, mangroves are
threatened by various pathogens all the time (Andrews, 1976; Binder,
Hibbett, Wang, & Farnham, 2006; Hyde, 1989; Jones, 2011; Maria &
Sridhar, 2002; Zhang et al., 2013). Among them, Cladosporium sp. may
pose a potential risk to mangroves. Thus, the potential pathogenicity
of Cladosporium spp. in mangrove environments was assessed in this
study. Three species of mangroves, namely Avicennia marina, Kandelia
candel and Rhizophora stylosa, which were dominant in Zhanjiang Bay,
were selected as hosts for experimentation.
2 | MATERIALS AND METHODS
2.1 | Sampling site
Data were collected on 09 Jan 2010 in a survey voyage of the
Zhanjiang Bay area (110°22.556′ E-­110°34.300′ E, 21°04.469 ‘N -­
21°16.165′ N). Sample sites (Figure 1) were located using a Garmin

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F I G U R E   1   Sampling stations in Zhanjiang Bay in winter 2009
GPS 76 system. The 19 sites were divided into seven cross sections:
A, B, C, D, E, F and G (Figure 1).
2.2 | Sample collection
Water samples were taken from the surface layer (0.5 m from the sur-
face) and bottom layer (0.5 m from the bottom) using HOM-­1 plastic
water sampling tanks. Water samples were transferred into 100-­mL
centrifuge tubes and then set on ice in styrofoam boxes. The samples
were returned to the laboratory and processed within 1 week.
Healthy mangrove leaves of Avicennia marina, Kandelia candel and
Rhizophora stylosa were collected from the same area.
2.3 | Isolation
Culture medium was prepared as described by Ronald (2004) with
slight modifications (100 g potato, 20 g agarose and 1000 mL natural
sea water). Fungi were isolated using the plating method, in which the
medium was supplemented with addition of 60 mg/L penicillin before
pouring the plates to inhibit bacteria (Raghukumar et al., 2004). Next,
100 μl sea water was poured on a plate and incubated at 28°C for
48 h. Hyphal tips were used to transfer isolates to potato dextrose
agar (PDA) dishes. Triplicates were made for each sample.
2.4 | Identification
2.4.1 | Morphological Identification
Isolates were spread on plates and incubated at 28°C for 48 h. The ap-
pearance, conidia and spores of the colony were examined for colour,
size and texture.
2.4.2 | rDNA ITS Sequencing
Genomic DNA was isolated as described by White, Bruns, Lee, and
Taylor (1990). ITS segments were amplified with ITS1 (5′-­TCCGTA
GGTGAACCTGCGG-­3′) and ITS4 primers (5′-­TCCTCCGCTTATTGA
TATGC-­3′). Amplification was performed in 25 μl containing 2.5 μl of
10 ×  buffer, 1 μl of 2.5 mmol/L dNTP, 1 μl of template DNA (30 ng),
1 μl of each primer (5 μmol), 1 μl of Taq DNA polymerase and 18 μl
of water. Thermal cycles were 2-­min preheating at 94°C, 35 cycles of
amplification (94°C for 30 s, 55°C for 30 s and 72°C for 1 min) and a
final extension for 10 min at 72°C. The temperature was finally held
at 4°C for 10 min. The rDNA ITS sequences of isolates were submit-
ted to GenBank, and the accession numbers were received. These
sequences were also confirmed using the basic local alignment search
tool (BLAST) database on the NCBI website (http://www.ncbi.nim.nih.
gov).
2.5 | Pathogenicity test
Healthy leaves were removed from the plants and wiped with
75% ethanol. The pathogenicity tests (inoculations) were then
performed with mycelial discs applied to the axial surfaces of the
leaves (IldikÓ, Tivadar, & Pe′ter, 2012; Liu & Xie, 2014; Sezer &
Dolar, 2012). Disposable needles were used to make five wounds
per inoculation. Three 5-­mm-­diameter discs were inoculated on
each leaf for K. candel and R. stylosa, while two discs were inocu-
lated on A. marina due to its small leaf size. Mycelial discs were cut
from fungal subcultures and grown for 7 days. Each isolate was
applied to three leaves, which were then placed in high humidity
conditions inside sealed plastic boxes containing moistened filter
paper. Fresh 5-­mm PDA discs were used as a control. All treat-
ments were incubated at 27°C for 5 days. The mycelial discs and
PDA agar discs were subsequently removed from the leaf to be
photographed. The test was repeated three times, giving a total of
nine times per isolate. Following incubation, the pathogens were
re-­isolated from diseased leaves onto fresh PDA containing strep-
tomycin. The resulting isolates were compared with the original
isolates used in the inoculations. The pathogenicity process was
considered complete when the test leaves became necrotic or
discoloured.
Prevalence = XY 100%; X: amount of pathogenic isolates; Y: total
test isolates.
3 | RESULTS
3.1 | Isolates and identification
Fungi were isolated from the Zhanjiang Bay area. Ninety-­one isolates
of Cladosporium sp. were obtained from seven different cross sec-
tions (Liu et al., 2013). The sequences were submitted to GenBank,
with ­accession numbers between JN098038 and JN098126 (Table 1).
Based on the combined morphological and molecular information, the
91 strains were identified as the following: 32 were C. sphaerospermum
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Penzig, which is characterized by secondary ramoconidia with
1-­septate and globose conidia and with 99% similarity to the acces-
sion NO.GU017501.1 by BLAST (Table 1 and Figure 2d) (Zalar et al.,
2007), 26 were C. cladosporioides (Fresen.) de Vries, which is charac-
terized by secondary ramoconidia aseptate, intercalary conidia not
rostrate and with 99% similarity to the accession NO.EF577236.1 by
BLAST (Table 1 and Figure 2a)(Bensch et al., 2010), 14 were C. coloca-
siae Sawada, which is characterized by conidia in short branched chains
and with 99% similarity to the accession NO.AF393694.2 by BLAST
(Table 1 and Figure 2g) and six were C. tenuissimum Cooke, which is
characterized by conidiophores nodulose with a head-­like swollen
apex and with 99% similarity to the accession NO.HM148211.1 by
BLAST (Table 1 and Figure 2e)(Bensch et al., 2010). In addition, three
of each of the following were identified: C. oxysporum Berkeley et
Curtis, which is characterized by conidiophores nodulose to nodose
with conidiogenous loci restricted to swellings and with 99% similarity
to the accession NO.KU216746.1 by BLAST, C. perangustum Bensch,
Crous & U. Braun, which is characterized by secondary ramoconidia
narrow and with 100% similarity to the accession NO.HM148147.1
by BLAST and C. uredinicola Spegazzini, which is characterized by
conidiophores with a tilt apex and with 99% similarity to the ac-
cession NO.AY362001.1 by BLAST representatively (Table 1 and

T A B L E   1   Isolates for pathogenicity tests

Cladosporium
Species sphaerospermum C. cladosporioides C. colocasiae C. tenuissimum C. oxysporum C. perangustum C. uredinicola

Isolate number 1D-­4,4S-­21,8S-­2, 8S-­1,8S-­3,8S-­7,8S-­8, 6D-­4,7S-­9,12D-­7, 4S-­6,6D-­3, 7S-­10 6D-­4 21D-­4

8S-­9,10S-­2,11D-­3, 9S-­2,11S-­4,11S-­5, 12D-­12,15D-­7,21S-­1 17S-­3
12D-­6,13S-­9,16S-­11, 11S-­9,15D-­5,15D-­8,
15D-­2,15D-­6,20S-­5, 20D-­6,20D-­7,23D-­2
20S-­6,21S-­2,21D-­5,
23D-­3
GenBank JN098044 JN098043 JN098058 JN098038 JN098042 JN098039 JN098123
accession no.
JN098050 JN098045 JN098059 JN098041
of isolates
JN098053 JN098046 JN098081 JN098058
JN098054 JN098047 JN098125
JN098051 JN098048
JN098057 JN098054
JN098079 JN098053
JN098088 JN098055
JN098120 JN098080
JN098121 JN098118
JN098124 JN098119
JN098132 JN098131
JN098126
Accession no. GU017501.1 EF577236.1 AF393694.2 HM148211.1 KU216746.1 HM148147.1 AY362001.1
by BLAST
Identity (%) 99 99 99 99 99 100 99

(a) (b) (c) (g)

F I G U R E   2   Cladosporium a: C. (d) (e) (f)

cladosporioides (8S-­1 JN098043); b:
C. oxysporum (7S-­10 JN098042); c:
C. perangustum (6D-­4 JN098039); d:
C. sphaerospermum (8S-­2 JN098044);
e: C. tenuissimum (6D-­3 JN098038);
f: C. uredinicola (21D-­4 JN098123);
g: C. colocasiae (21S-­1 JN098125)
Scale=10μm
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T A B L E   2   Pathogenicity tests of Cladosporium spp. on three mangroves (after 5d)

Prevalence(%)

Cladosporium
Species cladosporioides C. colocasiae C. oxysporum C. perangustum C. sphaerospermum C. tenuissimum C. uredinicola Total (%)

Number of test 13 7 1 1 16 3 1 42
strain (strains)
Avicennia marina 92.30 100.00 Pathogenic Pathogenic 93.75 100.00 Pathogenic 93.40
Kandelia candel 84.62 100.00 Pathogenic Pathogenic 87.50 66.67 Pathogenic 83.52
Rhizophora stylosa 7.69 28.57 None None None None None 7.41

(a) (b) (c)

(d) (e)

F I G U R E   3   Pathogenicity tests of
Cladosporium sp. (after 5d) a: K. candel by
20D-­6 (JN098118); (b and c): K. candel
by 20D-­6 (JN098118) on both sides; d:
A. marina by 8S-­1 (JN098043); e: R. stylosa
by 12D-­7 (JN098058). The image shows
the inoculated leaf (left) and, for contrast,
the leaf with PDA discs (right).

Figure 2b,c,f) (Bensch et al., 2010; Zalar et al., 2007). Finally, ten of
the strains could not be identified.
3.2 | Pathogenicity
Forty-­two of the 91 isolates were selected for pathogenicity tests of
Cladosporium sp. on mangrove leaves (Table 1). The results indicated
that the isolates could cause necrosis or discoloration of the mangrove
leaves, infecting A. marina and K. candel with a pathogenic prevalence
of 93.40% and 83.52%, respectively, and R. stylosa to a lesser extent
with a pathogenic prevalence of 7.41% (Table 2). A. marina was found
most vulnerable to the effects of pathogens, with a pathogenic rate
of 100% with C. colocasiae, 93.7% for C. sphaerospermum and 92.30%
for C. cladosporioides (Table 2). The vast majority of A. marina leaves
showed necrotic symptoms after inoculation (Figure 3d). The leaves
of K. candel were the second most vulnerable, with a pathogenic prev-
alence of 100% for C. colocasiae, 87.50% for C. sphaerospermum and
84.62% for C. cladosporioides. Most of the leaves of K. candel showed
necrotic or discoloration symptoms after inoculation (Figure 3a–c).
R. stylosa was infected by two isolates only, causing only slight discol-
oration in the leaves (Figure 3e).
4 | DISCUSSION
C. colocasiae had the strongest pathogenic effect towards A. marina
and K. candel, causing severe necrosis in the leaves. It was found path-
ogenic to the terrestrial taro plant; this initially appeared as olivaceous
leaf spots that progressed to tan to brown and finally to necrosis
(Holcomb, 1989). Our tests indicated that C. colocasiae could cause
severe necrosis in the leaves of mangroves in addition to the taro. This
species was also isolated from deep sea samples collected from South
China Sea (Zhang et al., 2013).
C. cladosporioides and C. sphaerospermum caused serious necrosis
in the leaves of A. marina and K. candel. Previously, the two species
were reported as common pathogens of terrestrial plants (Bensch
et al., 2010; Zalar et al., 2007). Nevertheless, this is the first study
to verify the pathogenic effect of the two species on A. marina and
K. candel.
After an examination of C. oxysporum, C. perangustum and C. te-
nuissimum, it was determined that C. uredinicola was pathogenic to
A. marina and K. candel. Although a lesser amount of each strain was
tested, a clear threat from the strains towards the two mangroves was
established.
LIUet al.
Most of Cladosporium isolates from the Zhanjiang Bay area could
have originated from terrestrial plants that were brought into the
ocean with the flow of the river.
In comparison among the three plants, A. marina was the most
susceptible to Cladosporium, K. candel was the second, while R. stylosa
was demonstrated the strongest resistance to this species.
To the best of our knowledge, this is the first report to verify the
pathogenic effects of Cladosporium on mangroves. The pathogenic-
ity tests indicated that the two mangrove species were significantly
damaged by Cladosporium. The results suggested that favourable en-
vironmental conditions would further advance the disease. Additional
concerns would be warranted under more severe levels of pollution.
ACKNOWLE DGE ME N TS
This research was supported by Doctoral Fund of Guangdong Ocean
University (E15037). We thank Prof. Jiang Zide from South China
Agricultural University, Guangzhou, China, for his helpful suggestions.
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