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Distribution and diversity of endophytes inseagrasses

Ambayeram VENKATACHALAMa, Nagamani THIRUNAVUKKARASUb,Trichur S. SURYANARAYANANa,*aVivekananda Institute of Tropical Mycology (VINSTROM), Ramakrishna Mission Vidyapith, Chennai 600004, IndiabPG & Research Department of Botany, Ramakrishna Mission Vivekananda College, Chennai 600004, India

a r t i c l e i n f o

Article history:

Received 21 March 2014

Revision received 26 June 2014

Accepted 17 July 2014

Available online

Corresponding editor:

Kevin Hyde

Keywords:

Endophytic fungi

Endosymbionts

Leaf

Marine angiosperms

Rhizome

* Corresponding author.E-mail address: t_sury2002@yahoo.com (T

http://dx.doi.org/10.1016/j.funeco.2014.07.0031754-5048/ª 2014 Elsevier Ltd and The Britis

a b s t r a c t

The rhizome and leaf tissues of 10 seagrass species (seven of the family Cymodoceaceae

and three of the family Hydrocharitaceae) collected along the coast of Tamilnadu state,

southern India were sampled for the presence of fungal endophytes. A culture-based study

revealed that the colonization frequency (CF%) of the endophytes was generally lower than

that reported for terrestrial plants and that members of Eurotiomycetes dominate the

endophyte assemblage in these marine angiosperms. The CF% of the endophytes was more

for the rhizome than for the leaves. Species of Aspergillus, Paecilomyces and Penicillium

occurred in high CF% and could be isolated from both the tissue types of seagrasses

belonging to both the families.

ª 2014 Elsevier Ltd and The British Mycological Society. All rights reserved.

Introduction (Govinda Rajulu et al., 2014). However, relatively less informa-

Although the symptomless endosymbionts of plants, the

endophytes, have been the focus of numerous studies, infor-

mation regarding various aspects of their biology is incomplete

(Suryanarayanan, 2013). The distribution and diversity of

endophytes of many terrestrial plant species and plant com-

munities have been investigated (Gazis et al., 2012; Li et al., 2012;

Govinda Rajulu et al., 2013). It is now well established that

endophytes of terrestrial and mangrove plants are a potential

source of a variety of novel bioactive metabolites

(Suryanarayanan et al., 2009; Kharwar et al., 2011; R€osenberg

et al., 2013; Debbab et al., 2013) and industrial enzymes

(Suryanarayanan et al., 2012; Nagarajan et al., 2014) and are

vested with attributes suited for technological exploitation

.S. Suryanarayanan).

h Mycological Society. Al

tion is available on endophytes of marine plants viz. the macro

algae and seagrasses. A few recent studies reveal that marine

macroalgae harbour endophytes exhibiting technologically

desirable properties (Zuccaro et al., 2008; Suryanarayanan et al.,

2010; Flewelling et al., 2013; Suryanarayanan and Johnson,

2014). Studies on endophyte association of seagrasses include

those of Newell and Fell (1980), Wilson (1998), Alva et al. (2002),

Devarajan et al. (2002), Sakayaroj et al. (2010), Mata and

Cebri�an (2013) and Shoemaker and Wyllie-Echeverria (2013).

Thesepertain tostudiesononeora fewspeciesofseagrasses.To

estimate the diversity of endophytes associated with these

marine angiosperms, we screened the leaf and rhizome tissues

of ten seagrass species growing along the coast of Tamilnadu

state of southern India.

l rights reserved.

Distribution and diversity of endophytes in seagrasses 61

Materials and methods

Collection site

The ten seagrass species Cymodocea serrulata (CS), Cymodocea

sp. 1 (CM), Cymodocea sp. 2 (CY), Halodule beaudettei (HW),

Halodule sp. (HL), Halodule uninervis (HN), Syringodium sp. 1 (SR)

belonging to the family Cymodoceaceae and Enhalus acoroides

(EA), Thalassia sp. 1 (TH) and Thalassia sp. 2 (TA) belonging to

the family Hydrocharitaceae were collected from Mallipatti-

nam, Keezhakkarai and Mandapam along the eastern coast of

Tamilnadu state, southern India. The plants were identified

based on morphological characters (Ramamurthy et al., 1992).

Healthy, mature plants having undamaged leaves and grow-

ing in shallow seawater were collected and brought to the

laboratory in sterile polythene bags and processed within

24 hr as follows.

Isolation of endophytes

The plants were washed thoroughly in running tap water and

air-dried. Tissue segments (0.5 cm2) were cut from the mid

portion of mature leaves and rhizomes and surface sterilized

as follows. Segments were rinsed in 70 % ethanol for 5 s,

immersed in 4 % sodium hypochlorite for 60 s and finally

rinsed in sterile distilled water (Devarajan et al., 2002). Seg-

ments thus prepared were placed on potato dextrose agar

medium amended with chloramphenicol (150 mg l�1). The

efficacy of the sterilization procedure was confirmed by the

method of Schulz et al. (1998). Ten segments were placed on

20ml growthmedium contained in a 9 cm diam Petri dish; 100

segments of leaf or rhizome were screened for each seagrass

species. The Petri dishes were sealed using Parafilm� and

incubated in a light chamber for 21 d (Bills and Polishook, 1992;

Suryanarayanan, 1992). The light regimen was 12 hr light

followed by 12 hr darkness. The light chamber had a bank of

three four foot Philips day light fluorescent lamps. The tissue

segments received 2200 lux of light through the Petri dish lid

as measured by Lutron (Germany) Lx-101 Lux meter. The

incubation temperature was 26 � 1 �C. The Petri dishes were

observed periodically and the endophytes growing out of the

segments were isolated in PDA slants and identified based on

culture characteristics. The sterile isolates that could not be

assigned to any taxonomic group were given codes using

culture characteristics such as growth rates, colony surface

textures and hyphal pigmentation (Bills and Polishook, 1994;

Dobranic et al., 1995). Such sterile forms were assumed to

represent different taxonomic species (Bills and Polishook,

1994). The Colonization Frequency (CF%) and occurrence of

Aspergillus spp. (ASP%) were calculated as follows (Hata and

Futai, 1995).

CF% ¼ Number of segments colonized by an endophyteTotal number of segments observed

� 100

ASP% ¼ Total number of Aspergillus spp: isolates in a tissue type of a seagrasTotal number of all isolates obtained from that tissue of the seagrass

A Jaccard Similarity Index (JI) was used to compare the

species composition of the leaf and rhizome tissues

(Magurran, 1988).

JI% ¼ caþ b� c

� 100

a ¼ Number of species in leaf assemblage

b ¼ Number of species in rhizome assemblage

c ¼ Number of species in common between the two

assemblages

A rarefaction curve was used to estimate the number of

species in a given sub-sample of isolates obtained from rhi-

zome or leaf (Koellner et al., 2004).

Results

No true marine fungus could be isolated but marine-derived

fungi were present as endophytes in seagrass tissues. The

rhizome tissue of all the seagrasses screened harboured

endophytes. Excepting CS and TA, the leaves of all the plants

had endophytes (Table 1). Among rhizome samples, the

maximum number (45) of endophyte was present in HW and

themaximumnumber (7) of endophyte species was recovered

from CY and TH. With leaf samples, the maximum number

(37) of isolate was present in EA; the maximum number (4) of

endophyte specieswas recovered CM, EA, HL andHN (Table 1).

Screening of 2000 tissue segments (1000 each of leaf and

rhizome) from 10 plant species yielded 305 endophyte isolates

representing 32 morphospecies (Table 1). A molecular

approach is necessary for discerning distinct species among

these morphospecies. From the rhizome and leaf tissues 173

and 132 isolateswere recovered indicating that rhizomeswere

more densely colonized. A Rarefaction analysis also showed

that the endophyte assemblage in rhizomes is more species

rich since 100 isolates from rhizomes would have 17 species

while thesamenumberof isolates fromleaveswouldhaveonly

13 species (Fig 1). Aspergillus terreus, (133 isolates, 43.6 %), Pae-

cilomyces sp.1 (59 isolates, 19.3%) and Penicillium sp. (32 isolates,

10.5%)were themost commonendophytespeciesandcouldbe

isolated from both rhizome and leaf of seagrass belonging to

both the families (Table 1).A. terreus could be isolated from leaf

or rhizome of all seagrasses except for TH. This fungus was

dominant in the leavesof EA,HLandHNand in the rhizomesof

CM,HWandHN(Table1). SpeciesofAspergilluswereharboured

by all the seagrass species and constituted 7e96 % of all the

isolates (Table 2). Apart from Aspergillus, Paecilomyces and Pen-

icillium spp., the other fungi were sparsely represented

(Table 1). A Jaccard Similarity index showed that species

composition of the endophyte assemblage of rhizome tissue

overlapped that of the leaf tissue by 29.6 %.

s� 100

Table 1 e CF% of endophytic fungi isolated from leaf and rhizome tissues of ten species of seagrasses. Abbreviations as in Table 2

Fungi CS CM CY HW HL HN SR TH TA EA

Leaf Rhizome Leaf Rhizome Leaf Rhizome Leaf Rhizome Leaf Rhizome Leaf Rhizome Leaf Rhizome Leaf Rhizome Leaf Rhizome Leaf Rhizome

Alternaria sp. 1

Aphanocladium sp. 2

Aspergillus niger 3 1 2 5

Aspergillus sp. 2 1 1 6

Aspergillus sp. 4 5

Aspergillus sp. 5 2

Aspergillus sp. 12 1

Aspergillus terreus 1 2 5 9 1 43 3 1 37 4 1 1 23 2

Chaetomium sp. 4 1

Chaetomium sp. 5 1

Cladosporium sp. 1 1 1 2 1 1 1

Cladosporium sp. 2 2

Coelomycete

form 1

1

Colletotrichum sp. 5 1

Curvularia sp. 1 5 1

Curvularia sp. 3 1

Curvularia

tuberculata

1

Fusarium sp. 2 3 1

Fusarium sp. 4 3

Fusarium sp. 7

Nigrospora sp. 1

Paecilomyces sp. 1 3 18 12 2 17 1 3 3

Paecilomyces sp. 2 10 3 3

Penicillium sp. 1 15 4 2 2 6

Phialophora sp. 1

Sterile form 1 1 1

Sterile form 2 1

Sterile form 6 1

Sterile form 11 2

Sterile form 14 1

Torula sp. 1

Total No. of

species

a 6 4 4 3 7 2 2 4 3 4 3 3 4 1 7 a 4 4 4

Total No. of

isolates

a 22 7 10 21 36 16 45 6 20 41 7 3 4 1 14 a 6 37 9

a No fungi isolated.

62

A.Venkatach

alam

etal.

0

5

10

15

20

25

0 20 40 60 80 100 120 140

E(Sn

)

Isolates (n)

Leaf Rhizome

Fig 1 e Rarefaction curves showing expected number

[E(Sn)] of endophyte species from leaf and rhizome of

seagrass species.

Distribution and diversity of endophytes in seagrasses 63

Discussion

The CF% of endophytes in all the species of seagrass screened

here was generally low when compared to those reported for

terrestrial plants. This observation is in consonance with

earlier reports on endophytes of different seagrass species

(Wilson, 1998; Alva et al., 2002; Devarajan et al., 2002;

Sakayaroj et al., 2010). Various reasons have been attributed

to explain this low frequency of endophyte infections in sea-

grasses. These include inhibition of endophytes of seagrass by

competing colonizers such as diatoms, and bacteria

(Devarajan et al., 2002; Rodriguez, 2008), the saline and oxygen

deficient environment of seagrasses presenting an unfav-

ourable condition for fungal colonization (Nielsen et al., 1999),

and the elaboration of defense chemicals by seagrass species

to ward off invading microbes (Ross et al., 2008). Furthermore,

endophytes (Kusari et al., 2013) including those of seagrasses

(Supaphon et al., 2013) produce antifungal compounds and

these may inhibit colonization by other fungal species as has

been observed for maize (Wicklow et al., 2005). It is possible

that these factors singly or in combination are responsible for

the low endophyte infections observed for seagrasses.

Table 2 e Percentage of Aspergillus spp. isolated asendophytes from leaf and rhizome tissues of seagrasses

Host Abbreviation % of Aspergillus spp.

Leaf Rhizome

Cymodocea serrulata CS a 14

Cymodocea sp. 1 CM 29 50

Cymodocea sp. 2 CY 43 17

Halodule beaudettei HW 0 96

Halodule sp. HL 67 15

Halodule uninervis HN 93 57

Syringodium sp. 1 SR 67 0

Thalassia sp. 1 TH 0 7

Thalassia sp. 2 TA a 50

Enhalus acoroides EA 92 22

a No endophytic fungi isolated from Cymodocea serrulata and Tha-

lassia sp. 2.

Although all the plants were collected from similar locations,

the leaves of CS and TA were free of endophytes. This indi-

cates the play of host factors in endophyte colonization of

seagrasses. The use of different surface sterilization protocols

and growth media as well as environmental PCR-based

endophyte detection of fungi, as has been attempted for ter-

restrial plants (Arnold et al., 2007), would reveal the real status

of endophyte association in seagrasses.

The higher CF% of endophytes in rhizome than in leaf

tissue could be due to the proximity of rhizomes to the sedi-

ment which would carry more fungal inoculum than the

seawater in which the leaf floats (Shoemaker and Wyllie-

Echeverria, 2013). According to these authors, the rhizome of

Zostera marina is more densely colonized by endophytes than

that of Phyllospadix scouleri because the latter grows on rocks

and has little sediment around it.

Species of Aspergillus and Penicillium dominate the endo-

phyte assemblages in several seagrass species occurring in

different parts of the world. These include Thalassia testudi-

num, Halodule bermudensis and Syringodium filiforme from Ber-

muda, USA (Wilson, 1998), T. testudinum, Z. marina and Z.

Japonica from Hong Kong and The Philippines (Alva et al.,

2002), Halophila ovalis from India (Devarajan et al., 2002), T.

testudinum from Puerto Rico (Rodriguez, 2008), Enhalus acor-

oides from Thailand (Sakayaroj et al., 2010), Halodule wrightii

and T. testudinum from Gulf of Mexico (Mata and Cebri�an,

2013) and Zostera marina, Z. japonica and Phyllospadix scouleri

from San Juan Archipelago, Washington State, USA

(Shoemaker and Wyllie-Echeverria, 2013). We now report a

Penicillium sp. dominates the endophyte assemblage in the

rhizome Cymodocea serrulata, and Aspergillus spp. dominate

the endophyte assemblages of the rhizomes of Cymodocea sp

1 and Halodule beaudettei and those of the leaves of Cymodocea

sp 2., Enhalus acoroides, Halodule sp., H. uninervis, and Syrin-

godium sp., occurring in India. Species of Aspergillus also

dominate the endophyte assemblages of many seaweeds

(Suryanarayanan et al., 2010) and are also recovered as

endosymbionts of marine sponges (Thirunavukkarasu et al.,

2012; Suryanarayanan, 2012a). Such a wide spread geo-

graphical distribution and expanded host range of these

marine-derived fungi indicates the wide ecological amplitude

and adaptations in these fungi. It is interesting to note that

while fungi belonging to Sordariomycetes, Dothidiomycetes

and Pezizomycetes dominate the endophyte assemblages of

most terrestrial plants (Suryanarayanan et al., 2011; U’Ren

et al., 2012), members of Eurotiomycetes dominate the

endophyte communities of fresh water (Sandberg et al., 2014)

and marine angiosperms. Studying the endophyte status of

floating and submerged leaves of heterophyllous hydro-

phytes might help understanding this apparent environment

specificity shown by these fungal groups. New generation

sequencing such as 454 pyrosequencing are essential to

explain the composition of endophyte assemblages in sea-

grasses (Toju et al., 2013).

It is pertinent to mention that, like the endophytes of ter-

restrial plants (Weber, 2009), the marine-derived fungal

endophytes of seagrasses produce many novel bioactive

metabolites of unprecedented structures (Bugni and Ireland,

2004; Geetha et al., 2011; Suryanarayanan, 2012b; Supaphon

et al., 2013) and sometimes only under saline conditions

64 A. Venkatachalam et al.

(Suryanarayanan, 2012b). This and the fact that seagrass

meadows are threatened globally due to various stressors

(Orth et al., 2006) warrant the need for a more concerted effort

to study endophyte association of these marine angiosperms.

Acknowledgements

Our thanks are due to the Secretary, Ramakrishna Mission

Vidyapith, Mylapore, Chennai for providing facilities. NT

thanks the Department of Science and Technology, Govern-

ment of India for funding the SERB project (SB/EMEQ-005/

2013).

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