Research ArticleEndophytic Fungal Diversity in Medicinal Plants ofWestern Ghats India

Monnanda Somaiah Nalini1 Ningaraju Sunayana2 and Harischandra Sripathy Prakash2

1 Department of Studies in Botany University of Mysore Manasagangotri Mysore Karnataka 570 006 India2Department of Studies in Biotechnology University of Mysore Manasagangotri Mysore Karnataka 570 006 India

Correspondence should be addressed to Monnanda Somaiah Nalini nmsomaiahgmailcom

Received 24 September 2013 Accepted 16 April 2014 Published 11 May 2014

Academic Editor Raeid Abed

Copyright copy 2014 Monnanda Somaiah Nalini et al This is an open access article distributed under the Creative CommonsAttribution License which permits unrestricted use distribution and reproduction in any medium provided the original work isproperly cited

Endophytes constitute an important component of microbial diversity and in the present investigation seven plant species withrich ethnobotanical uses representing six families were analyzed for the presence of endophytic fungi from their natural habitatsduring monsoon (MayJune) and winter (NovemberDecember) seasons of 2007 Fungal endophytes were isolated from healthyplant parts such as stem root rhizome and inflorescence employing standard isolation methods One thousand five hundred andtwenty-nine fungal isolates were obtained from 5200 fragments Stem fragments harbored more endophytes (8037) than roots(1922) 31 fungal taxa comprised of coelomycetes (65) hyphomycetes (32) and ascomycetes (3) Fusarium AcremoniumColletotrichum Chaetomium Myrothecium Phomopsis and Pestalotiopsis spp were commonly isolated Diversity indices differedsignificantly between the seasons (119875 lt 0001) Species richness was greater for monsoon isolations than winter Host specificity wasobserved for few fungal endophytes UPGMA cluster analysis grouped the endophytes into distinct clusters on the basis of geneticdistance This study is the first report on the diversity and host-specificity of endophytic fungal taxa were from the semi evergreenforest type in Talacauvery subcluster of Western Ghats

1 Introduction

The microbes residing in the internal parts of plant tissuescalled ldquoendophytesrdquo constitute a group of plant symbiontsand are a component of microbial diversity Endophytes offerplethora of unknown advantages to the host with immenseapplications in agriculture andmedicine [1 2] Recently chal-lenging hypotheses related to endophyte diversity [3] theirrole in oxidative stress protection [4] heavy metal tolerance[5] and as components of tropical community ecology [67] have emerged A perusal of the literature over the pastdecades indicated many ethnomedicinal plant species withrich botanical history sampled fromunique ecological nichesspecies are known to harbor potential endophytic microbes[8]

There has been an increasing surge of interest amongthe research groups for the isolation of endophytes from thetropical plant species [9 10] owing to high plant diversityOne such region represents the Western Ghats stretching

a length of 1600Km from the river Tapti in the state ofGujarat to the Southern tip of Kerala recognized as one of the34 hot spots of biodiversityTheWestern Ghats represent richflora with enormous species diversity as well as endemic taxaand are therefore recognized as one among the hot spots ofthe world [11] Western Ghats are divided into seven subclus-ters A proposal to include and declare 39 sites in this regionas the World Natural Heritage Cluster Site by UNESCO isunderway (httpwwwatreeorgwg unesco whs)

India has many regions of unique ecological nicheharboring variety of medicinal plants One such region inthe peninsular India is Kodagu District the land of coffeecultivation Kodagu is situated in the Western Ghats ofpeninsular India and is known for its majestic mountainranges coffee plantations and teak wood forests The Tala-cauvery subcluster (12∘171015840 to 12∘271015840Nand 75∘261015840 to 75∘331015840E) ofthe Western Ghats is situated in Kodagu The altitude rangesfrom 1525 above mean sea level Annual precipitation of3525mm is largely restricted during May to October

Hindawi Publishing CorporationInternational Journal of BiodiversityVolume 2014 Article ID 494213 9 pageshttpdxdoiorg1011552014494213

2 International Journal of Biodiversity

Table 1 Details of medicinal plants collected from the natural habitats of Talacauvery subcluster of Western Ghats

Plant specieslowast Common name Ayurvedic name Family Habit Parts collectedTylophora asthmatica(W and A) Indian ipecacuanha Anthrapachaka Asclepiadaceae Twiner Stem

Rubia cordifolia L Indian madder Majith Rubiaceae Climber StemPlumbago zeylanica L White leadwort Chitramool Plumbaginaceae Shrub StemPhyllanthus amarus(Schum andThonn) Niruri Bhoomyamalaki Euphorbiaceae Herb Stem and root

Eryngium foetidum L Fit weedspinyserrated coriander Bhandhanya Apiaceae Herb Roots and stemCentella asiatica L Asiatic pennywort Gotu Kola Apiaceae Runner Stolon roots and inflorescenceZingiber sp Wild ginger Shunti Zingiberaceae Herb Rhizome aerial stem and rootTen plantslowast were sampled pooled and used for isolation of endophytes [13]

although premonsoon showers are not uncommon duringFebruary to April The average temperature is 23∘C Kodaguhas a reservoir of forest belts and diverse vegetation rangingfrom tropical wet evergreen forests to scrub jungles Severaltribes residing in the forests still use medicinal plants ofethnopharmacological importance as the source of naturalmedication for their ailments [12] Ethnomedicinal plants areoften used in ayurvedic medicinal system in India for thetreatment of various diseases

Despite the reports of ethnomedicinal plants of thisregion the biodiversity and the endophytic microbes of thisregion remain unexplored Therefore in the present investi-gation seven medicinal plants representing six families weresubjected to diversity studies on fungal endophytes duringtwo seasons

2 Methodology

21 Plant Materials and Study Site Plant parts such as stemroot rhizome and inflorescence were collected from sevenhealthy medicinal plant species Tylophora asthmatica Rubiacordifolia Plumbago zeylanica Phyllanthus amarus Eryn-gium foetidum Centella asiatica and Zingiber sp inhabitingthe natural vegetation of the Talacauvery Region of WesternGhats located at 012∘171015840 to 012∘271015840N and 075∘261015840 to 075∘331015840Eof Kodagu Karnataka during the monsoon (May to June)and winter seasons (November-December) of 2007 (Table 1)The natural vegetation is an evergreensemievergreen type offorests The mean temperature was 23∘C and mean annualprecipitation is 3525mm Herbarium specimens of the plantswere prepared and submitted to the herbarium collectionsin the DOS in Botany University of Mysore Ten individualplants from eachwere pooled for isolationsThe samples wereplaced in polyethylene bags labeled transported in ice boxto the laboratory and placed in a refrigerator at 4∘C untilisolation All samples were processed within 24 h of collec-tion

22 Isolation and Identification of Endophytic Fungi Sampleswere washed thoroughly in distilled water blot dried andfirst immersed in 70 ethanol (vv) for one min followedby second immersion in sodium hypochlorite (35 vv) forthree minutes They were rinsed three times in changes of

sterile distilled water and dried on sterile blotters under theairflow to ensure complete drying Bits of 10 times 01 cm sizewere excised with the help of a sterile blade A total of 5200segments from stem roots inflorescence and rhizomes ofplant species were placed on water agar (25) supplementedwith the antibiotic streptomycin sulphate (100mgL) Fortysegments were plated per plate The plates were wrapped incleanwrap cling film and incubated at 22∘Cwith 12 h light anddark cycles for up to 6 to 8 weeksThe effectiveness of surfacesterilization of tissues was checked by placing the aliquots ofsterilants on agar plates and observing fungal colonies if anyfor two weeks [14]

Periodically the bits were examined for the appearanceof fungal colony and each colony that emerged from seg-ments was transferred to antibiotic-free potato dextrose agarmedium (PDA 2) to aid identification The morphologicalidentification of the isolates was done based on the fungalcolony morphology and characteristics of the reproductivestructures and spores [15ndash17] Sporulation was induced byinoculating cultures onto sterilized banana leaf bits (one cm2)impregnated on potato dextrose agar in petri dishes Allfungal mounts were made on microscopic glass slides inlactophenol-cotton blue and sealed with nail polish Cultureswhich failed to sporulate were grouped as mycelia steriliaAll the fungal isolates have been catalogued as DST serieswith plant code and maintained as culture collections of thedepartment by cryopreservation on PDA overlaid with 15glycerol (vv) at ndash20∘C in a deep freezer

23 Data Analysis Isolation rate (IR) the measure of fungalrichness of a sample was calculated as the number ofisolates obtained from tissue segments divided by the totalnumber of segments and expressed as fractions but not aspercentages [18] The colonization frequency (CF) expressedas percentage was calculated according to Kumaresan andSuryanarayanan [19] as follows

CF =

Number of tissue segments colonized by a fungusTotal number of tissue segments plated

times100

(1)

International Journal of Biodiversity 3

0

20

40

60

80

100

Stem Root Rhizome InflPlant parts

MonsoonWinter

Relat

ive i

sola

tion

()

Figure 1 Relative seasonal isolations of fungal endophytes fromplant parts of medicinal species

The percentage of dominant endophytes (119863) was calculatedbased on the CF divided by the total number of endophytestimes 100 [20]

Differences in the extent of colonization of the sam-ples were analyzed by univariant analysis of variance (one-way ANOVA) and Tukeyrsquos honestly significant difference(HSD) as post hoc test using the statistical softwareSPSS160 The fungal isolations were considered for anal-ysis of ANOVA and Tukeyrsquos HSD Simpson and Shannondiversity indices were calculated for endophytic fungifrom different seasons with Estimate 119878 software (version6 httpviceroyeebuconneduestimates) Species richnesswas calculated using the onlineweb page rarefactor calculator(httpwww2biologyualbertacajbrzustorarefactphp)

Rarefaction indices were employed to compare thespecies richness among the plant species during two seasonsThe expected number of species in 119873 isolations was calcu-lated [21] Unweighted pair group method with arithmeticmean (UPGMA) cluster analysis was applied for all theisolates from plant species based on the number of isolatesrecovered from each plant species using a dendrogram con-structed based on Neirsquos genetic distances [22] using tools forpopulation genetics analysis (TFPGA) software [23]

3 Results

A total of 1529 isolates were obtained from 5200 tissuefragments from seven medicinal plant species The extentof endophytes colonization varied in plant parts with stemfragments harboring 80 of endophytic isolates followed byroot (1922) In other plant parts colonization was lowerIsolations of endophytes from various plant parts showedgreater numbers of endophytes during monsoon than winter(Figure 1) The high isolation rates (IR) of fungal endophyteswere recorded as 141 to 158 for T asthmatica in bothseasons while in Zingiber sp low rates of isolations

TA HA PHY RC EF PZ ZN

Rate

of i

sola

tion

Medicinal plants

MonsoonWinter

2

18

16

14

12

1

08

06

04

02

0

Figure 2 Seasonal isolation rates of fungal endophytes frommedic-inal species Endophytic fungi were isolated from seven medicinalplant species during monsoon and winter seasons respectively

were obtained (Figure 2) Thirty-one fungal taxa wereidentified which consisted of coelomycetes (65)hyphomycetes (32) and ascomycete isolations of 3The frequency of fungal colonization (CF) differedamong the seven plant species (Table 2) Fusarium spAcremonium Chaetomium and Phoma are some of theendophytes with high colonization frequency The dominantfungal genera include Fusarium spp (119863 = 1064) andAcremonium (119863 = 948) Few endophytic fungi such asA strictum had wide distributions in host plants and wereisolated from most plants with the exception of Zingibersp and Plumbago zeylanica whereas species of FusariumTrichoderma Curvularia and Penicillium were isolated frommore than three plant species

Host-specificity was observed for few of the fungalendophytes isolated from two of the seven medicinal plants(Table 2) Colletotrichum dematium Nigrospora oryzaeHeinesia rubi Pestalotiopsis guepinii and unidentified redpycnidial form were isolated from the stem segments of Tasthmatica only while in Rubia cordifolia one endophyticPericonia exhibited specificity P islandicum and T viridewere isolated from root segments of Phyllanthus amarus

Diversity indices of fungal endophytes variedwithin plantspecies as well as between seasons (Table 3) High Shannon-Weiner diversity index was recorded for T asthmatica (1198671 =260) and P amarus (1198671 = 227) during monsoon and winterseasons whereas low indices were recorded for E foetidumand Zingiber during monsoon and winter seasons respec-tively 42 of the total 31 taxa were found inmonsoon seasonwhile 55 of them colonized in both seasons Simpson index(1119897) was high for T asthmatica with a richness of 19 fungalspecies during monsoon season while P amarus recordedhighest richness of species during winter season Rarefac-tion curves calculated for the endophytic fungal isolationsindicated maximum species richness for T asthmatica and Pamarus during monsoon and winter isolations respectively

4 International Journal of Biodiversity

Table2Colon

izationfre

quencylowast

ofendo

phyticfung

iisolated

from

plantp

artsof

sevenmedicinalplantspecies

Season

sMon

soon

Winter

Plantspecies

TARC

PPH

YEF

CAZN

TARC

PZPH

YEF

CAZN

119863(

)PlantP

arts

Stem

SR

SR

ISt

RI

SRh

RStem

SR

SR

StR

IS

RhR

Endo

phytes

Acremonium

strictum

250

160

mdash100

9080

90mdash

20

90mdash

mdashmdash

mdash125

75150

mdash50

25

60

50

35

mdashmdash

mdashmdash

948

Alternariaalternata

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash85

mdashmdash

mdash15

mdashmdash

mdashmdash

mdashmdash

mdash065

Aspergillus

terreus

115

mdash50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash70

mdashmdash

mdashmdash

mdashmdash

mdashmdash

154

Redpycnidia

50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

033

Botryodiplodiatheobrom

ae25

75mdash

25

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash50

mdash25

mdashmdash

15mdash

mdashmdash

mdashmdash

mdash14

1Cladosporiu

mherbarum

mdashmdash

mdash25

mdashmdash

mdashmdash

mdashmdash

mdashmdash

05

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

020

Colletotrichum

dematium

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

200

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash13

1C

lindemuthianum

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash160

mdashmdash

mdashmdash

100

35

mdashmdash

mdashmdash

60

mdashmdash

mdashmdash

mdashmdash

232

Chaetomium

globosum

275

mdashmdash

mdashmdash

mdash160

mdashmdash

mdashmdash

mdash10

mdash160

mdash35

mdashmdash

50

mdashmdash

mdashmdash

mdashmdash

mdash451

Curvularialunata

mdashmdash

mdash25

mdashmdash

mdashmdash

60

40

mdashmdash

mdashmdash

mdash100

mdash75

mdashmdash

mdashmdash

25

mdashmdash

mdashmdash

212

Fusariu

msp

mdashmdash

mdashmdash

15mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

15mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

020

Fgram

inearum

mdashmdash

mdashmdash

mdashmdash

mdashmdash

95mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash10

mdashmdash

mdashmdash

mdash069

Foxysporum

75200

60

mdashmdash

mdashmdash

1035

mdashmdash

mdashmdash

05

mdash15

mdashmdash

mdashmdash

1040

mdashmdash

mdash10

mdash301

Fsolani

50

100

mdashmdash

45

mdash50

mdashmdash

100

mdashmdash

mdash40

mdashmdash

mdash45

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash281

Fpseudonygamai

mdashmdash

mdashmdash

mdash25

mdashmdash

mdashmdash

mdashmdash

mdash05

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash020

Fverticilloides

210

mdash55

100

mdash75

mdashmdash

mdashmdash

20

mdashmdash

mdashmdash

110

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash373

Gela

sinospora

spp

25

mdashmdash

20

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

029

Hein

esiarubi

15mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash010

Mem

noniellaspp

85

25

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash069

Myrotheciu

mverrucaria

30

mdash75

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

60

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash10

8Nigrospora

oryzae

50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

033

Peric

oniasp

mdash175

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash114

Pesta

lotio

psisguepinii

20

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

013

Pesta

lotio

psissp

mdashmdash

mdash50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash20

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash059

Phom

aspp

60

100

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

90mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash16

3Ph

omopsis

sp

50

mdashmdash

mdashmdash

mdashmdash

mdash10

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash039

Penicilliu

msp

100

45

mdashmdash

20

mdash10

mdashmdash

30

mdashmdash

mdashmdash

mdash20

50

25

mdashmdash

mdash15

mdashmdash

mdashmdash

206

Pislandicum

mdashmdash

mdash75

10mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

055

Trich

odermaharzianu

mmdash

mdash75

mdashmdash

45

30

mdashmdash

mdashmdash

mdash15

mdashmdash

mdashmdash

50

15mdash

mdashmdash

50

mdashmdash

20

mdash19

6Tvirid

emdash

mdashmdash

mdash50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

10mdash

mdashmdash

mdashmdash

mdashmdash

mdash039

Sphaeronem

asp

25

mdashmdash

120

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

082

Verticillium

albo-atru

mmdash

mdash35

mdashmdash

mdashmdash

mdashmdash

mdashmdash

10mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash029

Pycnidialforms

mdash75

150

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

147

Sterile

mycelia

60

mdash35

50

mdash10

mdashmdash

mdashmdash

mdashmdash

mdash20

mdashmdash

45

mdashmdash

mdashmdash

mdashmdash

mdashmdash

144

Total

314

191

107

126

4647

682

4479

06

610

143

7263

7334

1829

2025

00

60

1529

314

191

107

172

117

123

22143

7263

107

4745

61529

lowast

200segm

entswerep

lated

forfrequ

ency

analysisdu

ringmon

soon

andwinterseasonsT

ATylophora

asthmatica

RC

RubiacordifoliaP

ZPlum

bago

zeylanica

PHY

Phyllanthu

samarusE

FEryngium

foetidum

CA

CentellaasiaticaZN

ZingiberspSstemStsto

lon

RrootR

hrhizom

eIinflo

rescence

International Journal of Biodiversity 5

Table 3 Diversity indices (1198671) and species richness of the medicinal plants

Monsoon Winter

Plant species Total isolates Total species richness Diversity indices Total isolates Total species richness Diversity indicesSimpson Shannon Simpson Shannon

Tylophora asthmatica 314 19 090 260 143 70 078 166Rubia cordifolia 191 9 086 205 72 60 079 162Plumbago zeylanica 107 8 084 196 63 4 063 114Phyllanthus amarus 172 14 087 229 107 110 089 227Eryngium foetidum 117 9 077 168 47 6 075 153Centella asiatica 123 8 083 188 45 6 076 157Zingiber sp 22 8 079 181 6 2 044 064

20

18

16

14

12

10

8

6

4

2

01 25 50 74 95 120 140 165 190

Number of isolations

TARCPZPHY

EFCAZN

Monsoon

Expe

cted

num

ber o

f spe

cies

(E(s))

Figure 3 Rarefaction curvesof fungal endophytes from monsoonisolations (number of isolations versus expected number of speciesE(119904)

) TA Tylophora asthmatica RC Rubia cordifolia PZ Plumbagozeylanica PHY Phyllanthus amarus EF Eryngium foetidum CACentella asiatica and ZN Zingiber sp

(Figures 3 and 4) Differences in the number of isolates andcolonization frequency differed significantly between seasons(119875 lt 0001) as indicated in Table 4

Neirsquos genetic distance between endophytes isolated fromplant species ranged from 03185 (between populations ofZingiber and C asiatica) to 09116 (between populationsof Zingiber and T asthmatica) (Table 5) which was widelyranged This indicates a closer relationship between endo-phytic fungal patterns of both plants In general fungalspecies from T asthmatica is most distanced from the other

TARCPZPHY

EFCAZN

Winter

12

10

8

6

4

2

01 7 13 19 25 31 37 43 49 55

Number of isolations

Expe

cted

num

ber o

f spe

cies

(E(s))

Figure 4 Rarefaction curves of fungal endophytes from winterisolations (number of isolations versus expected number of speciesE(119904)

) TA Tylophora asthmatica RC Rubia cordifolia PZ Plumbagozeylanica PHY Phyllanthus amarus EF Eryngium foetidum CACentella asiatica and ZN Zingiber sp

plants studied In order to represent the relationships amongplant species cluster analysis (UPGMA)was used to generatea dendrogram based on Neirsquos genetic distances betweenpopulations (Figure 5) In this dendrogram all plants forma distinct cluster When the transect line was placed atapproximately 04 on the distance scale two distinct groupswere formed The first cluster was formed by CA-ZN while

6 International Journal of Biodiversity

Table 4 ANOVA table of seasonal variation of endophytic fungi analyzed from seven medicinal plant species

Sum of squares Degrees of freedom Mean2119865 Significance

Between seasons 119319 26 458919 34382 0001Within seasons 1001062 75 13346 mdash mdashTotal 1293296 101 mdash mdash mdash

Table 5 Neirsquos genetic distance of plant species analyzed for endo-phytic fungi

TA RC PZ PHY EF CA ZNTA lowastlowastlowast 07885 05008 06061 04055 07885 09116RC lowastlowastlowast 05008 07239 04055 03610 06061PZ lowastlowastlowast 06633 03610 05008 04520PHY lowastlowastlowast 04520 05008 06633EF lowastlowastlowast 04055 03610CA lowastlowastlowast 03185ZN lowastlowastlowast

lowastIndicates that genetic distance between the same genus does not exist it isshown for different genera

0700 0525 0350 0175 0000

CA

ZN

PZ

EF

RC

PHY

TA

Figure 5 UPGMA cluster analysis of fungal endophytes represent-ing seven medicinal plant species Plant codes are TA Tylophoraasthmatica RC Rubia cordifolia PZ Plumbago zeylanica PHYPhyllanthus amarus EF Eryngium foetidum CA Centella asiaticaand ZN Zingiber sp

PZ-EF formed the second cluster PHY RC and TA are foundoutside the cluster

4 Discussion

Medicinal plants are considered as a repository of ldquoendo-phytic microbesrdquo living in the internal tissues of plants Thequest for identifying novel bioactives from the endophyticfungi has resulted in the sampling of host plants such asherbs shrubs tree species and vines in unique places ofecological adaptations around the rainforests of the worldSuch niches harbor great species diversity unintervened by

human activities Efforts in this direction to sample plantslocated in the rainforests around the world with potentialethnomedicinal values have resulted in the isolation of fungalendophytes unique to a particular plant species with distinctbioactivity

41 Endophyte Colonization inMedicinal Species Themedic-inal plant species were sampled from the Talacauvery sub-cluster situated in the Kodagu District of Western Ghats ofSouthern India This region is among one of the 34 hot spotsof biodiversity Recently a proposal to include this biodiver-sity spot in the list of UNESCOHeritage cluster site is under-way (httpwwwatreeorgwg unesco whs) The natives aswell as the ethnic tribes inhabiting this region still dependon the plants as a source of medicine to cure some of theailments [12] Seven medicinal plant species assigned to sixplant families were selected for the study in natural popula-tions in two seasons from a single location from the studyarea stretching over an area of 25 kilometers Sampling wasconducted during monsoon and winter seasons as two ofthe herbaceous species E foetidum and Zingiber sp growonly till the second half of the year (June to December) andtheir nonavailability during summer (March toMay)makes itdifficult to consider the summer season for endophyticanalysis

From 5200 segments of plant materials a total of 1529isolates were obtained these were grouped into 31 taxaMycelia sterilia the fungal taxa that failed to sporulate werealso reported from this study This fungal group is prevalentin endophytic studies [24] The fungal endophytes wereanalyzed from four plant parts namely stem root rhizomeand inflorescence however their occurrence in root andinflorescence was investigated for few plant species only asthe phenology and sampling of plants never correlated withseasons The leaves were not considered for isolations sincesome of the plants were climbers and stragglers with delicatehairy surfaces and stringent surface sterilization techniqueswould render them unsuitable for plating on agar mediumRelative percentages of endophytic isolations from stemsegments were greater (8037) than isolations from roots(1922) Our results are supported by the earlier work ofHuang et al [25] on 29 traditional Chinese medicinal plantsthat fungal endophytes aremore frequent in stem tissues thanroots Among the fungal taxa coelomycete isolations weremore dominant than hyphomycetes and have been found inearlier studies in endophytes of tree species [26]

Endophytes such asColletotrichum PhomaAcremoniumChaetomium Botryodiplodia and Trichoderma were isolatedwith 119863 gt 20 Few fungal taxa that are less frequently iso-lated are Pestalotiopsis Penicillium islandicum Cladosporiumherbarum Alternaria alternata F graminearum Phomopsis

International Journal of Biodiversity 7

and Sphaeronema Colletotrichum spp are the most fre-quently encountered endophytic fungi [18] whereas Pestalo-tiopsis spp are well documented as endophytes of many rain-forest plants [27 28] tropical tree species namelyTerminaliaarjuna [29] Azadirachta indica [30] and many herbs andshrubs [25 31 32] It is necessary to screen newer plant speciesfor the isolation of fungal endophytes as Hawksworth andRossman [33] estimate that there are still millions of speciesof fungi yet to be identified Differences in the colonizationfrequencies of endophytes during two seasons were observedand more isolation during monsoon season is due to the factthat the slimy conidia of fungal spores are dispersed betterby rain splashes and germination of conidia is influenced byclimatic factors [34]

42 Host-Specificity of Fungal Endophytes We observed thatsome fungal taxa exhibited host-specificity a phenomenonoften associated with endophytes Three plant species Tasthmatica R cordifolia and P amarus were host-specificto endophytes The red pycnidial endophyte (TA-005) wasisolated from the stem fragments of T asthmatica onlysuggesting the host-specificity of this endophyte Pestalo-tiopsis guepinii was isolated from the stem segments of Tasthmatica It has been reported as an endophyte ofWollemianobilis growing in Sydney Australia [35] Heinesia rubiP islandicum and TA-005 are new reports of fungi asendophytes Host-specificity of endophytic fungi has beenobserved earlier for grasses [36] orchids [37] and forest treespecies [38 39] Recently Sun et al [40] reiterated the termldquohost-specificityrdquo as taxa that occur exclusively on a statedhost but not on other hosts in the same habitat [41] Ourstudies also indicate the host-specificity of endophytes as theplant species were sampled from a single habitat

43 Seasonal Diversity of Fungal Endophytes Diversityindices for fungal endophytes as analyzed by Shannon-Weiner (1198671) and Simpson (1119897) indices indicated differencesin seasonal variation and species richness High indices werenoted for T asthmatica (1198671 = 26) and P amarus (1198671 = 227)during monsoon and winter seasons respectivelyThe fungalspecies did not differ significantly between plant specieswhereas they differed between seasons (119875 lt 0001) Seasonalvariation in fungal isolates and colonization frequency hasbeen reported for many host plants [42 43] High coloniza-tion frequency as well as the species richness of endophyticfungi is limited to leaf segments rather than stem or bark seg-ments of host plants sampled from five medicinal species ofKudremukh Region of Western Ghats [44] Species richnessin our study is limited to stem fragments among the plantparts considered for analysis

Most studies on fungal endophytes in tropics haverevealed remarkable patterns of endophyte colonizationand estimates of diversity in foliages of forest tree speciesrepresenting various sites such as Panamanian Forest [45]and Iwokrama Forest Reserve Guyana [39] In the NilgiriBiosphere Reserve Western Ghats India 75 dicotyledonousspecies in three different tropical forest types were sampledto study foliar endophytes and diversity [10] The endophyte

diversity in forest types was limited due to loose hostaffiliations among endophytes Studies on foliar endophytesfrom the sampling of herbaceous and shrubby medicinalplant species have revealed differences in the colonizationrates as well as seasonal diversity inMalnad Region of BhadraWildlife Sanctuary in Southern India [32 46]

The present study provides firsthand information onthe diversity and seasonal influence on the colonizationfrequencies of endophytic fungi from selected medicinalplants from one of the subclusters of biodiversity hot spotsin the Western Ghats of Southern India Although theisolation and analysis of endophyte communities in herbsshrubs and trees are not uncommon each of the studies isunique with reference to number of hosts species of fungalendophytes and their specificityThe fungal endophytes havebeen subjected to fermentation studies and extracts are beingtested for biological activities

5 Conclusion

The study provides firsthand information on the diversityand seasonal influence on the colonization frequencies ofendophytic fungi from sevenmedicinal plants fromone of thesubclusters of biodiversity hot spots in the Western Ghats ofSouthern IndiaThe present investigation is the first isolationof endophytes from the medicinal species and their plantpartsThough isolation of endophytes has been accomplishedfrom various forest types and locations around the globeeach study is unique in documenting newer endophytic taxaWe are currently working on the fermentation of fungalendophytes to obtain newer antioxidants with therapeuticapplications

Conflict of Interests

The authors declare no conflict of interests regarding thepublication of this paper

Acknowledgments

This work was carried out with the financial assistancefrom the Department of Science amp Technology (DST)-SERCDivision Government of India under the Women Scien-tist Scheme [DST-WOS (A)] awarded to Monnanda Soma-iah Nalini (DST sanction no SRWOS (A)LS-762006 dt02082007) Monnanda Somaiah Nalini is thankful to theChairman DOS in Biotechnology for providing the neces-sary facilities for the completion of the project

References

[1] K Clay J Holah and J A Rudgers ldquoHerbivores cause a rapidincrease in hereditary symbiosis and alter plant communitycompositionrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 102 no 35 pp 12465ndash124702005

[2] P Alvarez-Loayza J F White Jr M S Torres et al ldquoLight con-verts endosymbiotic fungus to pathogen influencing seedling

8 International Journal of Biodiversity

survival and niche-space filling of a common tropical treeIriartea deltoideardquo PloS ONE vol 6 no 1 2011

[3] R Linnakoski H Puhakka-tarvainen andA Pappinen ldquoEndo-phytic fungi isolated from Khaya anthotheca in Ghanardquo FungalEcology vol 5 no 3 pp 298ndash308 2012

[4] J F White Jr and M S Torres ldquoIs plant endophyte-mediateddefensive mutualism the result of oxidative stress protectionrdquoPhysiologia Plantarum vol 138 no 4 pp 440ndash446 2010

[5] H-Y Li D-W Li C-M He Z-P Zhou T Mei and H-MXu ldquoDiversity and heavy metal tolerance of endophytic fungifrom six dominant plant species in a Pb-Zn mine wasteland inChinardquo Fungal Ecology vol 5 no 3 pp 309ndash315 2012

[6] A E Arnold ldquoUnderstanding the diversity of foliar endo-phytic fungi progress challenges and frontiersrdquo Fungal BiologyReviews vol 21 no 2-3 pp 51ndash66 2007

[7] K D Hyde and K Soytong ldquoThe fungal endophyte dilemmardquoFungal Diversity vol 33 pp 163ndash173 2008

[8] G Strobel and B Daisy ldquoBioprospecting for microbial endo-phytes and their natural productsrdquoMicrobiology and MolecularBiology Reviews vol 67 no 4 pp 491ndash502 2003

[9] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007

[10] T S Suryanarayanan T S Murali N Thirunavukkarasu M BGovinda Rajulu G Venkatesan and R Sukumar ldquoEndophyticfungal communities in woody perennials of three tropical foresttypes of the Western Ghats southern Indiardquo Biodiversity andConservation vol 20 no 5 pp 913ndash928 2011

[11] R A Mittermeier N Myers P R Gil and C G MittermeierHotspots Earthrsquos Biologically Richest and Most Endangered Ter-restrial Ecoregions Cemex Conservation International Wash-ington DC USA 2000

[12] R D Kshirsagar and N P Singh ldquoSome less known eth-nomedicinal uses from Mysore and Coorg districts Karnatakastate Indiardquo Journal of Ethnopharmacology vol 75 no 2-3 pp231ndash238 2001

[13] N YoganarasimhanMedicinal Plants of India Karnataka Inter-line Publishing Bangalore India 1996

[14] B Schulz U Wanke S Draeger and H J Aust ldquoEndophytesfrom herbaceous plants and shrubs effectiveness of surfacesterilization methodsrdquo Mycological Research vol 97 no 12 pp1447ndash1450 1993

[15] H Barnett and B Hunter Illustrated Genera of Imperfect FungiBurgess Publishing Minneapolis Minn USA 1998

[16] K H DomschW Gams and T AndersonCompendium of SoilFungi Academic Press New York NY USA 2003

[17] J F Leslie and B A Summerell The Fusarium LaboratoryManual Blackwell Publishing London UK 2006

[18] W Photita P W J Taylor R Ford K D Hyde and S Lumy-ong ldquoMorphological and molecular characterization of Col-letotrichum species from herbaceous plants inThailandrdquo FungalDiversity vol 18 pp 117ndash133 2005

[19] V Kumaresan and T S Suryanarayanan ldquoOccurrence and dis-tribution of endophytic fungi in a mangrove communityrdquoMycological Research vol 105 no 11 pp 1388ndash1391 2001

[20] V Kumaresan and T S Suryanarayanan ldquoEndophyte assem-blages in young mature and senescent leaves of Rhizophora

apiculata evidence for the role of endophytes inmangrove litterdegradationrdquo Fungal Diversity vol 9 pp 81ndash91 2002

[21] J A Ludwig and J F Reynolds Statistical Ecology A Primer onMethods and Computing John Wiley amp Sons New York NYUSA 1988

[22] M Nei ldquoGenetic distance between populationsrdquo AmericanNaturalist vol 106 no 949 pp 283ndash293 1972

[23] M P Miller Tools For Population Genetic Analyses (TFPGA)V L3 A Windows program For the Analysis of Allozyme AndmoleculAr Genetic data Department of Biological SciencesNorthern Arizona University Flagstaff Ariz USA 1997

[24] D C Lacap K D Hyde and E C Y Liew ldquoAn evaluationof the fungal rsquomorphotypersquo concept based on ribosomal DNAsequencesrdquo Fungal Diversity vol 12 pp 53ndash66 2003

[25] W Y Huang Y Z Cai K D Hyde H Corke and M SunldquoBiodiversity of endophytic fungi associated with 29 traditionalChinese medicinal plantsrdquo Fungal Diversity vol 33 pp 61ndash752008

[26] M V Tejesvi B Mahesh M S Nalini et al ldquoFungal endophyteassemblages from ethnopharmaceutically important medicinaltreesrdquo Canadian Journal of Microbiology vol 52 no 5 pp 427ndash435 2006

[27] N Raj Coelomycetous Anamorphs With Appendage BearingConidia Edwards Brothers Ann Harbor Michigan 1993

[28] G A Strobel ldquoMicrobial gifts from rain forestsrdquo CanadianJournal of Plant Pathology vol 24 no 1 pp 14ndash20 2002

[29] M V Tejesvi B Mahesh M S Nalini et al ldquoEndophytic fungalassemblages from inner bark and twig of Terminalia arjunaW amp A (Combretaceae)rdquo World Journal of Microbiology andBiotechnology vol 21 no 8-9 pp 1535ndash1540 2005

[30] B Mahesh M V Tejesvi M S Nalini et al ldquoEndophyticmycoflora of inner bark of Azadirachta indica A Jussrdquo CurrentScience vol 88 no 2 pp 218ndash219 2005

[31] K Rajagopal S Kalavathy S Kokila et al ldquoDiversity of fungalendophytes in few medicinal herbs of southern Indiardquo AsianJournal of Experimental Biological Sciences vol 1 pp 415ndash4182010

[32] Y L Krishnamurthy S B Naik and S Jayaram ldquoFungal com-munities in herbaceous medicinal plants from the malnadregion Southern IndiardquoMicrobes and Environments vol 23 no1 pp 24ndash28 2008

[33] D L Hawksworth and A Y Rossman ldquoWhere are all theundescribed fungirdquo Phytopathology vol 87 no 9 pp 888ndash8911997

[34] D Wilson and G C CarrolL ldquoInfection studies of Disculaquercina an endophyte of Quercus garryanardquo Mycologia vol86 no 5 pp 635ndash647 1994

[35] G A Strobel W M Hess J-Y Li et al ldquoPestalotiopsis guepiniia taxol-producing endophyte of the wollemi pine Wollemianobilisrdquo Australian Journal of Botany vol 45 no 6 pp 1073ndash1082 1997

[36] K Clay ldquoFungal endophytes of grassesrdquo Annual Review ofEcology and Systematics vol 21 no 1 pp 275ndash297 1990

[37] P Bayman L L Lebron R L Tremblay and D J Lodge ldquoVari-ation in endophytic fungi from roots and leaves of Lepanthes(Orchidaceae)rdquoNewPhytologist vol 135 no 1 pp 143ndash149 1997

[38] A EArnold ZMaynard andG SGilbert ldquoFungal endophytesin dicotyledonous neotropical trees patterns of abundance and

International Journal of Biodiversity 9

diversityrdquo Mycological Research vol 105 no 12 pp 1502ndash15072001

[39] P F Cannon andCM Simmons ldquoDiversity andhost preferenceof leaf endophytic fungi in the Iwokrama Forest ReserveGuyanardquoMycologia vol 94 no 2 pp 210ndash220 2002

[40] X Sun Q Ding K D Hyde and L D Guo ldquoCommunitystructure and preference of endophytic fungi of three woodyplants in a mixed forestrdquo Fungal Ecology vol 5 no 5 pp 624ndash632 2012

[41] P HollidayADictionary of Plant Pathology CambridgeUniver-sity Press Cambridge UK 1998

[42] J Collado G Platas and F Pelaez ldquoIdentification of an endo-phytic Nodulisporium sp from Quercus ilex in central Spainas the anamorph of Biscogniauxia mediterranea by rDNAsequence analysis and effect of different ecological factors ondistribution of the fungusrdquo Mycologia vol 93 no 5 pp 875ndash886 2001

[43] X-X Gao H Zhou D-Y Xu C-H Yu Y-Q Chen and L-HQu ldquoHigh diversity of endophytic fungi from the pharmaceuti-cal plant Heterosmilax japonica Kunth revealed by cultivation-independent approachrdquo FEMS Microbiology Letters vol 249no 2 pp 255ndash266 2005

[44] N S Raviraja ldquoFungal endophytes in five medicinal plantspecies from Kudremukh Range Western Ghats of IndiardquoJournal of Basic Microbiology vol 45 no 3 pp 230ndash235 2005

[45] A E Arnold Z Maynard G S Gilbert P D Coley and T AKursar ldquoAre tropical fungal endophytes hyperdiverserdquo EcologyLetters vol 3 no 4 pp 267ndash274 2000

[46] B S Naik J Shashikala and Y L Krishnamurthy ldquoDiversityof fungal endophytes in shrubby medicinal plants of Malnadregion Western Ghats Southern Indiardquo Fungal Ecology vol 1no 2-3 pp 89ndash93 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

2 International Journal of Biodiversity

Table 1 Details of medicinal plants collected from the natural habitats of Talacauvery subcluster of Western Ghats

Plant specieslowast Common name Ayurvedic name Family Habit Parts collectedTylophora asthmatica(W and A) Indian ipecacuanha Anthrapachaka Asclepiadaceae Twiner Stem

Rubia cordifolia L Indian madder Majith Rubiaceae Climber StemPlumbago zeylanica L White leadwort Chitramool Plumbaginaceae Shrub StemPhyllanthus amarus(Schum andThonn) Niruri Bhoomyamalaki Euphorbiaceae Herb Stem and root

Eryngium foetidum L Fit weedspinyserrated coriander Bhandhanya Apiaceae Herb Roots and stemCentella asiatica L Asiatic pennywort Gotu Kola Apiaceae Runner Stolon roots and inflorescenceZingiber sp Wild ginger Shunti Zingiberaceae Herb Rhizome aerial stem and rootTen plantslowast were sampled pooled and used for isolation of endophytes [13]

although premonsoon showers are not uncommon duringFebruary to April The average temperature is 23∘C Kodaguhas a reservoir of forest belts and diverse vegetation rangingfrom tropical wet evergreen forests to scrub jungles Severaltribes residing in the forests still use medicinal plants ofethnopharmacological importance as the source of naturalmedication for their ailments [12] Ethnomedicinal plants areoften used in ayurvedic medicinal system in India for thetreatment of various diseases

Despite the reports of ethnomedicinal plants of thisregion the biodiversity and the endophytic microbes of thisregion remain unexplored Therefore in the present investi-gation seven medicinal plants representing six families weresubjected to diversity studies on fungal endophytes duringtwo seasons

2 Methodology

21 Plant Materials and Study Site Plant parts such as stemroot rhizome and inflorescence were collected from sevenhealthy medicinal plant species Tylophora asthmatica Rubiacordifolia Plumbago zeylanica Phyllanthus amarus Eryn-gium foetidum Centella asiatica and Zingiber sp inhabitingthe natural vegetation of the Talacauvery Region of WesternGhats located at 012∘171015840 to 012∘271015840N and 075∘261015840 to 075∘331015840Eof Kodagu Karnataka during the monsoon (May to June)and winter seasons (November-December) of 2007 (Table 1)The natural vegetation is an evergreensemievergreen type offorests The mean temperature was 23∘C and mean annualprecipitation is 3525mm Herbarium specimens of the plantswere prepared and submitted to the herbarium collectionsin the DOS in Botany University of Mysore Ten individualplants from eachwere pooled for isolationsThe samples wereplaced in polyethylene bags labeled transported in ice boxto the laboratory and placed in a refrigerator at 4∘C untilisolation All samples were processed within 24 h of collec-tion

22 Isolation and Identification of Endophytic Fungi Sampleswere washed thoroughly in distilled water blot dried andfirst immersed in 70 ethanol (vv) for one min followedby second immersion in sodium hypochlorite (35 vv) forthree minutes They were rinsed three times in changes of

sterile distilled water and dried on sterile blotters under theairflow to ensure complete drying Bits of 10 times 01 cm sizewere excised with the help of a sterile blade A total of 5200segments from stem roots inflorescence and rhizomes ofplant species were placed on water agar (25) supplementedwith the antibiotic streptomycin sulphate (100mgL) Fortysegments were plated per plate The plates were wrapped incleanwrap cling film and incubated at 22∘Cwith 12 h light anddark cycles for up to 6 to 8 weeksThe effectiveness of surfacesterilization of tissues was checked by placing the aliquots ofsterilants on agar plates and observing fungal colonies if anyfor two weeks [14]

Periodically the bits were examined for the appearanceof fungal colony and each colony that emerged from seg-ments was transferred to antibiotic-free potato dextrose agarmedium (PDA 2) to aid identification The morphologicalidentification of the isolates was done based on the fungalcolony morphology and characteristics of the reproductivestructures and spores [15ndash17] Sporulation was induced byinoculating cultures onto sterilized banana leaf bits (one cm2)impregnated on potato dextrose agar in petri dishes Allfungal mounts were made on microscopic glass slides inlactophenol-cotton blue and sealed with nail polish Cultureswhich failed to sporulate were grouped as mycelia steriliaAll the fungal isolates have been catalogued as DST serieswith plant code and maintained as culture collections of thedepartment by cryopreservation on PDA overlaid with 15glycerol (vv) at ndash20∘C in a deep freezer

23 Data Analysis Isolation rate (IR) the measure of fungalrichness of a sample was calculated as the number ofisolates obtained from tissue segments divided by the totalnumber of segments and expressed as fractions but not aspercentages [18] The colonization frequency (CF) expressedas percentage was calculated according to Kumaresan andSuryanarayanan [19] as follows

CF =

Number of tissue segments colonized by a fungusTotal number of tissue segments plated

times100

(1)

International Journal of Biodiversity 3

0

20

40

60

80

100

Stem Root Rhizome InflPlant parts

MonsoonWinter

Relat

ive i

sola

tion

()

Figure 1 Relative seasonal isolations of fungal endophytes fromplant parts of medicinal species

The percentage of dominant endophytes (119863) was calculatedbased on the CF divided by the total number of endophytestimes 100 [20]

Differences in the extent of colonization of the sam-ples were analyzed by univariant analysis of variance (one-way ANOVA) and Tukeyrsquos honestly significant difference(HSD) as post hoc test using the statistical softwareSPSS160 The fungal isolations were considered for anal-ysis of ANOVA and Tukeyrsquos HSD Simpson and Shannondiversity indices were calculated for endophytic fungifrom different seasons with Estimate 119878 software (version6 httpviceroyeebuconneduestimates) Species richnesswas calculated using the onlineweb page rarefactor calculator(httpwww2biologyualbertacajbrzustorarefactphp)

Rarefaction indices were employed to compare thespecies richness among the plant species during two seasonsThe expected number of species in 119873 isolations was calcu-lated [21] Unweighted pair group method with arithmeticmean (UPGMA) cluster analysis was applied for all theisolates from plant species based on the number of isolatesrecovered from each plant species using a dendrogram con-structed based on Neirsquos genetic distances [22] using tools forpopulation genetics analysis (TFPGA) software [23]

3 Results

A total of 1529 isolates were obtained from 5200 tissuefragments from seven medicinal plant species The extentof endophytes colonization varied in plant parts with stemfragments harboring 80 of endophytic isolates followed byroot (1922) In other plant parts colonization was lowerIsolations of endophytes from various plant parts showedgreater numbers of endophytes during monsoon than winter(Figure 1) The high isolation rates (IR) of fungal endophyteswere recorded as 141 to 158 for T asthmatica in bothseasons while in Zingiber sp low rates of isolations

TA HA PHY RC EF PZ ZN

Rate

of i

sola

tion

Medicinal plants

MonsoonWinter

2

18

16

14

12

1

08

06

04

02

0

Figure 2 Seasonal isolation rates of fungal endophytes frommedic-inal species Endophytic fungi were isolated from seven medicinalplant species during monsoon and winter seasons respectively

were obtained (Figure 2) Thirty-one fungal taxa wereidentified which consisted of coelomycetes (65)hyphomycetes (32) and ascomycete isolations of 3The frequency of fungal colonization (CF) differedamong the seven plant species (Table 2) Fusarium spAcremonium Chaetomium and Phoma are some of theendophytes with high colonization frequency The dominantfungal genera include Fusarium spp (119863 = 1064) andAcremonium (119863 = 948) Few endophytic fungi such asA strictum had wide distributions in host plants and wereisolated from most plants with the exception of Zingibersp and Plumbago zeylanica whereas species of FusariumTrichoderma Curvularia and Penicillium were isolated frommore than three plant species

Host-specificity was observed for few of the fungalendophytes isolated from two of the seven medicinal plants(Table 2) Colletotrichum dematium Nigrospora oryzaeHeinesia rubi Pestalotiopsis guepinii and unidentified redpycnidial form were isolated from the stem segments of Tasthmatica only while in Rubia cordifolia one endophyticPericonia exhibited specificity P islandicum and T viridewere isolated from root segments of Phyllanthus amarus

Diversity indices of fungal endophytes variedwithin plantspecies as well as between seasons (Table 3) High Shannon-Weiner diversity index was recorded for T asthmatica (1198671 =260) and P amarus (1198671 = 227) during monsoon and winterseasons whereas low indices were recorded for E foetidumand Zingiber during monsoon and winter seasons respec-tively 42 of the total 31 taxa were found inmonsoon seasonwhile 55 of them colonized in both seasons Simpson index(1119897) was high for T asthmatica with a richness of 19 fungalspecies during monsoon season while P amarus recordedhighest richness of species during winter season Rarefac-tion curves calculated for the endophytic fungal isolationsindicated maximum species richness for T asthmatica and Pamarus during monsoon and winter isolations respectively

4 International Journal of Biodiversity

Table2Colon

izationfre

quencylowast

ofendo

phyticfung

iisolated

from

plantp

artsof

sevenmedicinalplantspecies

Season

sMon

soon

Winter

Plantspecies

TARC

PPH

YEF

CAZN

TARC

PZPH

YEF

CAZN

119863(

)PlantP

arts

Stem

SR

SR

ISt

RI

SRh

RStem

SR

SR

StR

IS

RhR

Endo

phytes

Acremonium

strictum

250

160

mdash100

9080

90mdash

20

90mdash

mdashmdash

mdash125

75150

mdash50

25

60

50

35

mdashmdash

mdashmdash

948

Alternariaalternata

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash85

mdashmdash

mdash15

mdashmdash

mdashmdash

mdashmdash

mdash065

Aspergillus

terreus

115

mdash50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash70

mdashmdash

mdashmdash

mdashmdash

mdashmdash

154

Redpycnidia

50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

033

Botryodiplodiatheobrom

ae25

75mdash

25

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash50

mdash25

mdashmdash

15mdash

mdashmdash

mdashmdash

mdash14

1Cladosporiu

mherbarum

mdashmdash

mdash25

mdashmdash

mdashmdash

mdashmdash

mdashmdash

05

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

020

Colletotrichum

dematium

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

200

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash13

1C

lindemuthianum

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash160

mdashmdash

mdashmdash

100

35

mdashmdash

mdashmdash

60

mdashmdash

mdashmdash

mdashmdash

232

Chaetomium

globosum

275

mdashmdash

mdashmdash

mdash160

mdashmdash

mdashmdash

mdash10

mdash160

mdash35

mdashmdash

50

mdashmdash

mdashmdash

mdashmdash

mdash451

Curvularialunata

mdashmdash

mdash25

mdashmdash

mdashmdash

60

40

mdashmdash

mdashmdash

mdash100

mdash75

mdashmdash

mdashmdash

25

mdashmdash

mdashmdash

212

Fusariu

msp

mdashmdash

mdashmdash

15mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

15mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

020

Fgram

inearum

mdashmdash

mdashmdash

mdashmdash

mdashmdash

95mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash10

mdashmdash

mdashmdash

mdash069

Foxysporum

75200

60

mdashmdash

mdashmdash

1035

mdashmdash

mdashmdash

05

mdash15

mdashmdash

mdashmdash

1040

mdashmdash

mdash10

mdash301

Fsolani

50

100

mdashmdash

45

mdash50

mdashmdash

100

mdashmdash

mdash40

mdashmdash

mdash45

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash281

Fpseudonygamai

mdashmdash

mdashmdash

mdash25

mdashmdash

mdashmdash

mdashmdash

mdash05

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash020

Fverticilloides

210

mdash55

100

mdash75

mdashmdash

mdashmdash

20

mdashmdash

mdashmdash

110

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash373

Gela

sinospora

spp

25

mdashmdash

20

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

029

Hein

esiarubi

15mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash010

Mem

noniellaspp

85

25

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash069

Myrotheciu

mverrucaria

30

mdash75

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

60

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash10

8Nigrospora

oryzae

50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

033

Peric

oniasp

mdash175

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash114

Pesta

lotio

psisguepinii

20

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

013

Pesta

lotio

psissp

mdashmdash

mdash50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash20

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash059

Phom

aspp

60

100

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

90mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash16

3Ph

omopsis

sp

50

mdashmdash

mdashmdash

mdashmdash

mdash10

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash039

Penicilliu

msp

100

45

mdashmdash

20

mdash10

mdashmdash

30

mdashmdash

mdashmdash

mdash20

50

25

mdashmdash

mdash15

mdashmdash

mdashmdash

206

Pislandicum

mdashmdash

mdash75

10mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

055

Trich

odermaharzianu

mmdash

mdash75

mdashmdash

45

30

mdashmdash

mdashmdash

mdash15

mdashmdash

mdashmdash

50

15mdash

mdashmdash

50

mdashmdash

20

mdash19

6Tvirid

emdash

mdashmdash

mdash50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

10mdash

mdashmdash

mdashmdash

mdashmdash

mdash039

Sphaeronem

asp

25

mdashmdash

120

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

082

Verticillium

albo-atru

mmdash

mdash35

mdashmdash

mdashmdash

mdashmdash

mdashmdash

10mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash029

Pycnidialforms

mdash75

150

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

147

Sterile

mycelia

60

mdash35

50

mdash10

mdashmdash

mdashmdash

mdashmdash

mdash20

mdashmdash

45

mdashmdash

mdashmdash

mdashmdash

mdashmdash

144

Total

314

191

107

126

4647

682

4479

06

610

143

7263

7334

1829

2025

00

60

1529

314

191

107

172

117

123

22143

7263

107

4745

61529

lowast

200segm

entswerep

lated

forfrequ

ency

analysisdu

ringmon

soon

andwinterseasonsT

ATylophora

asthmatica

RC

RubiacordifoliaP

ZPlum

bago

zeylanica

PHY

Phyllanthu

samarusE

FEryngium

foetidum

CA

CentellaasiaticaZN

ZingiberspSstemStsto

lon

RrootR

hrhizom

eIinflo

rescence

International Journal of Biodiversity 5

Table 3 Diversity indices (1198671) and species richness of the medicinal plants

Monsoon Winter

Plant species Total isolates Total species richness Diversity indices Total isolates Total species richness Diversity indicesSimpson Shannon Simpson Shannon

Tylophora asthmatica 314 19 090 260 143 70 078 166Rubia cordifolia 191 9 086 205 72 60 079 162Plumbago zeylanica 107 8 084 196 63 4 063 114Phyllanthus amarus 172 14 087 229 107 110 089 227Eryngium foetidum 117 9 077 168 47 6 075 153Centella asiatica 123 8 083 188 45 6 076 157Zingiber sp 22 8 079 181 6 2 044 064

20

18

16

14

12

10

8

6

4

2

01 25 50 74 95 120 140 165 190

Number of isolations

TARCPZPHY

EFCAZN

Monsoon

Expe

cted

num

ber o

f spe

cies

(E(s))

Figure 3 Rarefaction curvesof fungal endophytes from monsoonisolations (number of isolations versus expected number of speciesE(119904)

) TA Tylophora asthmatica RC Rubia cordifolia PZ Plumbagozeylanica PHY Phyllanthus amarus EF Eryngium foetidum CACentella asiatica and ZN Zingiber sp

(Figures 3 and 4) Differences in the number of isolates andcolonization frequency differed significantly between seasons(119875 lt 0001) as indicated in Table 4

Neirsquos genetic distance between endophytes isolated fromplant species ranged from 03185 (between populations ofZingiber and C asiatica) to 09116 (between populationsof Zingiber and T asthmatica) (Table 5) which was widelyranged This indicates a closer relationship between endo-phytic fungal patterns of both plants In general fungalspecies from T asthmatica is most distanced from the other

TARCPZPHY

EFCAZN

Winter

12

10

8

6

4

2

01 7 13 19 25 31 37 43 49 55

Number of isolations

Expe

cted

num

ber o

f spe

cies

(E(s))

Figure 4 Rarefaction curves of fungal endophytes from winterisolations (number of isolations versus expected number of speciesE(119904)

) TA Tylophora asthmatica RC Rubia cordifolia PZ Plumbagozeylanica PHY Phyllanthus amarus EF Eryngium foetidum CACentella asiatica and ZN Zingiber sp

plants studied In order to represent the relationships amongplant species cluster analysis (UPGMA)was used to generatea dendrogram based on Neirsquos genetic distances betweenpopulations (Figure 5) In this dendrogram all plants forma distinct cluster When the transect line was placed atapproximately 04 on the distance scale two distinct groupswere formed The first cluster was formed by CA-ZN while

6 International Journal of Biodiversity

Table 4 ANOVA table of seasonal variation of endophytic fungi analyzed from seven medicinal plant species

Sum of squares Degrees of freedom Mean2119865 Significance

Between seasons 119319 26 458919 34382 0001Within seasons 1001062 75 13346 mdash mdashTotal 1293296 101 mdash mdash mdash

Table 5 Neirsquos genetic distance of plant species analyzed for endo-phytic fungi

TA RC PZ PHY EF CA ZNTA lowastlowastlowast 07885 05008 06061 04055 07885 09116RC lowastlowastlowast 05008 07239 04055 03610 06061PZ lowastlowastlowast 06633 03610 05008 04520PHY lowastlowastlowast 04520 05008 06633EF lowastlowastlowast 04055 03610CA lowastlowastlowast 03185ZN lowastlowastlowast

lowastIndicates that genetic distance between the same genus does not exist it isshown for different genera

0700 0525 0350 0175 0000

CA

ZN

PZ

EF

RC

PHY

TA

Figure 5 UPGMA cluster analysis of fungal endophytes represent-ing seven medicinal plant species Plant codes are TA Tylophoraasthmatica RC Rubia cordifolia PZ Plumbago zeylanica PHYPhyllanthus amarus EF Eryngium foetidum CA Centella asiaticaand ZN Zingiber sp

PZ-EF formed the second cluster PHY RC and TA are foundoutside the cluster

4 Discussion

Medicinal plants are considered as a repository of ldquoendo-phytic microbesrdquo living in the internal tissues of plants Thequest for identifying novel bioactives from the endophyticfungi has resulted in the sampling of host plants such asherbs shrubs tree species and vines in unique places ofecological adaptations around the rainforests of the worldSuch niches harbor great species diversity unintervened by

human activities Efforts in this direction to sample plantslocated in the rainforests around the world with potentialethnomedicinal values have resulted in the isolation of fungalendophytes unique to a particular plant species with distinctbioactivity

41 Endophyte Colonization inMedicinal Species Themedic-inal plant species were sampled from the Talacauvery sub-cluster situated in the Kodagu District of Western Ghats ofSouthern India This region is among one of the 34 hot spotsof biodiversity Recently a proposal to include this biodiver-sity spot in the list of UNESCOHeritage cluster site is under-way (httpwwwatreeorgwg unesco whs) The natives aswell as the ethnic tribes inhabiting this region still dependon the plants as a source of medicine to cure some of theailments [12] Seven medicinal plant species assigned to sixplant families were selected for the study in natural popula-tions in two seasons from a single location from the studyarea stretching over an area of 25 kilometers Sampling wasconducted during monsoon and winter seasons as two ofthe herbaceous species E foetidum and Zingiber sp growonly till the second half of the year (June to December) andtheir nonavailability during summer (March toMay)makes itdifficult to consider the summer season for endophyticanalysis

From 5200 segments of plant materials a total of 1529isolates were obtained these were grouped into 31 taxaMycelia sterilia the fungal taxa that failed to sporulate werealso reported from this study This fungal group is prevalentin endophytic studies [24] The fungal endophytes wereanalyzed from four plant parts namely stem root rhizomeand inflorescence however their occurrence in root andinflorescence was investigated for few plant species only asthe phenology and sampling of plants never correlated withseasons The leaves were not considered for isolations sincesome of the plants were climbers and stragglers with delicatehairy surfaces and stringent surface sterilization techniqueswould render them unsuitable for plating on agar mediumRelative percentages of endophytic isolations from stemsegments were greater (8037) than isolations from roots(1922) Our results are supported by the earlier work ofHuang et al [25] on 29 traditional Chinese medicinal plantsthat fungal endophytes aremore frequent in stem tissues thanroots Among the fungal taxa coelomycete isolations weremore dominant than hyphomycetes and have been found inearlier studies in endophytes of tree species [26]

Endophytes such asColletotrichum PhomaAcremoniumChaetomium Botryodiplodia and Trichoderma were isolatedwith 119863 gt 20 Few fungal taxa that are less frequently iso-lated are Pestalotiopsis Penicillium islandicum Cladosporiumherbarum Alternaria alternata F graminearum Phomopsis

International Journal of Biodiversity 7

and Sphaeronema Colletotrichum spp are the most fre-quently encountered endophytic fungi [18] whereas Pestalo-tiopsis spp are well documented as endophytes of many rain-forest plants [27 28] tropical tree species namelyTerminaliaarjuna [29] Azadirachta indica [30] and many herbs andshrubs [25 31 32] It is necessary to screen newer plant speciesfor the isolation of fungal endophytes as Hawksworth andRossman [33] estimate that there are still millions of speciesof fungi yet to be identified Differences in the colonizationfrequencies of endophytes during two seasons were observedand more isolation during monsoon season is due to the factthat the slimy conidia of fungal spores are dispersed betterby rain splashes and germination of conidia is influenced byclimatic factors [34]

42 Host-Specificity of Fungal Endophytes We observed thatsome fungal taxa exhibited host-specificity a phenomenonoften associated with endophytes Three plant species Tasthmatica R cordifolia and P amarus were host-specificto endophytes The red pycnidial endophyte (TA-005) wasisolated from the stem fragments of T asthmatica onlysuggesting the host-specificity of this endophyte Pestalo-tiopsis guepinii was isolated from the stem segments of Tasthmatica It has been reported as an endophyte ofWollemianobilis growing in Sydney Australia [35] Heinesia rubiP islandicum and TA-005 are new reports of fungi asendophytes Host-specificity of endophytic fungi has beenobserved earlier for grasses [36] orchids [37] and forest treespecies [38 39] Recently Sun et al [40] reiterated the termldquohost-specificityrdquo as taxa that occur exclusively on a statedhost but not on other hosts in the same habitat [41] Ourstudies also indicate the host-specificity of endophytes as theplant species were sampled from a single habitat

43 Seasonal Diversity of Fungal Endophytes Diversityindices for fungal endophytes as analyzed by Shannon-Weiner (1198671) and Simpson (1119897) indices indicated differencesin seasonal variation and species richness High indices werenoted for T asthmatica (1198671 = 26) and P amarus (1198671 = 227)during monsoon and winter seasons respectivelyThe fungalspecies did not differ significantly between plant specieswhereas they differed between seasons (119875 lt 0001) Seasonalvariation in fungal isolates and colonization frequency hasbeen reported for many host plants [42 43] High coloniza-tion frequency as well as the species richness of endophyticfungi is limited to leaf segments rather than stem or bark seg-ments of host plants sampled from five medicinal species ofKudremukh Region of Western Ghats [44] Species richnessin our study is limited to stem fragments among the plantparts considered for analysis

Most studies on fungal endophytes in tropics haverevealed remarkable patterns of endophyte colonizationand estimates of diversity in foliages of forest tree speciesrepresenting various sites such as Panamanian Forest [45]and Iwokrama Forest Reserve Guyana [39] In the NilgiriBiosphere Reserve Western Ghats India 75 dicotyledonousspecies in three different tropical forest types were sampledto study foliar endophytes and diversity [10] The endophyte

diversity in forest types was limited due to loose hostaffiliations among endophytes Studies on foliar endophytesfrom the sampling of herbaceous and shrubby medicinalplant species have revealed differences in the colonizationrates as well as seasonal diversity inMalnad Region of BhadraWildlife Sanctuary in Southern India [32 46]

The present study provides firsthand information onthe diversity and seasonal influence on the colonizationfrequencies of endophytic fungi from selected medicinalplants from one of the subclusters of biodiversity hot spotsin the Western Ghats of Southern India Although theisolation and analysis of endophyte communities in herbsshrubs and trees are not uncommon each of the studies isunique with reference to number of hosts species of fungalendophytes and their specificityThe fungal endophytes havebeen subjected to fermentation studies and extracts are beingtested for biological activities

5 Conclusion

The study provides firsthand information on the diversityand seasonal influence on the colonization frequencies ofendophytic fungi from sevenmedicinal plants fromone of thesubclusters of biodiversity hot spots in the Western Ghats ofSouthern IndiaThe present investigation is the first isolationof endophytes from the medicinal species and their plantpartsThough isolation of endophytes has been accomplishedfrom various forest types and locations around the globeeach study is unique in documenting newer endophytic taxaWe are currently working on the fermentation of fungalendophytes to obtain newer antioxidants with therapeuticapplications

Conflict of Interests

The authors declare no conflict of interests regarding thepublication of this paper

Acknowledgments

This work was carried out with the financial assistancefrom the Department of Science amp Technology (DST)-SERCDivision Government of India under the Women Scien-tist Scheme [DST-WOS (A)] awarded to Monnanda Soma-iah Nalini (DST sanction no SRWOS (A)LS-762006 dt02082007) Monnanda Somaiah Nalini is thankful to theChairman DOS in Biotechnology for providing the neces-sary facilities for the completion of the project

References

[1] K Clay J Holah and J A Rudgers ldquoHerbivores cause a rapidincrease in hereditary symbiosis and alter plant communitycompositionrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 102 no 35 pp 12465ndash124702005

[2] P Alvarez-Loayza J F White Jr M S Torres et al ldquoLight con-verts endosymbiotic fungus to pathogen influencing seedling

8 International Journal of Biodiversity

survival and niche-space filling of a common tropical treeIriartea deltoideardquo PloS ONE vol 6 no 1 2011

[3] R Linnakoski H Puhakka-tarvainen andA Pappinen ldquoEndo-phytic fungi isolated from Khaya anthotheca in Ghanardquo FungalEcology vol 5 no 3 pp 298ndash308 2012

[4] J F White Jr and M S Torres ldquoIs plant endophyte-mediateddefensive mutualism the result of oxidative stress protectionrdquoPhysiologia Plantarum vol 138 no 4 pp 440ndash446 2010

[5] H-Y Li D-W Li C-M He Z-P Zhou T Mei and H-MXu ldquoDiversity and heavy metal tolerance of endophytic fungifrom six dominant plant species in a Pb-Zn mine wasteland inChinardquo Fungal Ecology vol 5 no 3 pp 309ndash315 2012

[6] A E Arnold ldquoUnderstanding the diversity of foliar endo-phytic fungi progress challenges and frontiersrdquo Fungal BiologyReviews vol 21 no 2-3 pp 51ndash66 2007

[7] K D Hyde and K Soytong ldquoThe fungal endophyte dilemmardquoFungal Diversity vol 33 pp 163ndash173 2008

[8] G Strobel and B Daisy ldquoBioprospecting for microbial endo-phytes and their natural productsrdquoMicrobiology and MolecularBiology Reviews vol 67 no 4 pp 491ndash502 2003

[9] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007

[10] T S Suryanarayanan T S Murali N Thirunavukkarasu M BGovinda Rajulu G Venkatesan and R Sukumar ldquoEndophyticfungal communities in woody perennials of three tropical foresttypes of the Western Ghats southern Indiardquo Biodiversity andConservation vol 20 no 5 pp 913ndash928 2011

[11] R A Mittermeier N Myers P R Gil and C G MittermeierHotspots Earthrsquos Biologically Richest and Most Endangered Ter-restrial Ecoregions Cemex Conservation International Wash-ington DC USA 2000

[12] R D Kshirsagar and N P Singh ldquoSome less known eth-nomedicinal uses from Mysore and Coorg districts Karnatakastate Indiardquo Journal of Ethnopharmacology vol 75 no 2-3 pp231ndash238 2001

[13] N YoganarasimhanMedicinal Plants of India Karnataka Inter-line Publishing Bangalore India 1996

[14] B Schulz U Wanke S Draeger and H J Aust ldquoEndophytesfrom herbaceous plants and shrubs effectiveness of surfacesterilization methodsrdquo Mycological Research vol 97 no 12 pp1447ndash1450 1993

[15] H Barnett and B Hunter Illustrated Genera of Imperfect FungiBurgess Publishing Minneapolis Minn USA 1998

[16] K H DomschW Gams and T AndersonCompendium of SoilFungi Academic Press New York NY USA 2003

[17] J F Leslie and B A Summerell The Fusarium LaboratoryManual Blackwell Publishing London UK 2006

[18] W Photita P W J Taylor R Ford K D Hyde and S Lumy-ong ldquoMorphological and molecular characterization of Col-letotrichum species from herbaceous plants inThailandrdquo FungalDiversity vol 18 pp 117ndash133 2005

[19] V Kumaresan and T S Suryanarayanan ldquoOccurrence and dis-tribution of endophytic fungi in a mangrove communityrdquoMycological Research vol 105 no 11 pp 1388ndash1391 2001

[20] V Kumaresan and T S Suryanarayanan ldquoEndophyte assem-blages in young mature and senescent leaves of Rhizophora

apiculata evidence for the role of endophytes inmangrove litterdegradationrdquo Fungal Diversity vol 9 pp 81ndash91 2002

[21] J A Ludwig and J F Reynolds Statistical Ecology A Primer onMethods and Computing John Wiley amp Sons New York NYUSA 1988

[22] M Nei ldquoGenetic distance between populationsrdquo AmericanNaturalist vol 106 no 949 pp 283ndash293 1972

[23] M P Miller Tools For Population Genetic Analyses (TFPGA)V L3 A Windows program For the Analysis of Allozyme AndmoleculAr Genetic data Department of Biological SciencesNorthern Arizona University Flagstaff Ariz USA 1997

[24] D C Lacap K D Hyde and E C Y Liew ldquoAn evaluationof the fungal rsquomorphotypersquo concept based on ribosomal DNAsequencesrdquo Fungal Diversity vol 12 pp 53ndash66 2003

[25] W Y Huang Y Z Cai K D Hyde H Corke and M SunldquoBiodiversity of endophytic fungi associated with 29 traditionalChinese medicinal plantsrdquo Fungal Diversity vol 33 pp 61ndash752008

[26] M V Tejesvi B Mahesh M S Nalini et al ldquoFungal endophyteassemblages from ethnopharmaceutically important medicinaltreesrdquo Canadian Journal of Microbiology vol 52 no 5 pp 427ndash435 2006

[27] N Raj Coelomycetous Anamorphs With Appendage BearingConidia Edwards Brothers Ann Harbor Michigan 1993

[28] G A Strobel ldquoMicrobial gifts from rain forestsrdquo CanadianJournal of Plant Pathology vol 24 no 1 pp 14ndash20 2002

[29] M V Tejesvi B Mahesh M S Nalini et al ldquoEndophytic fungalassemblages from inner bark and twig of Terminalia arjunaW amp A (Combretaceae)rdquo World Journal of Microbiology andBiotechnology vol 21 no 8-9 pp 1535ndash1540 2005

[30] B Mahesh M V Tejesvi M S Nalini et al ldquoEndophyticmycoflora of inner bark of Azadirachta indica A Jussrdquo CurrentScience vol 88 no 2 pp 218ndash219 2005

[31] K Rajagopal S Kalavathy S Kokila et al ldquoDiversity of fungalendophytes in few medicinal herbs of southern Indiardquo AsianJournal of Experimental Biological Sciences vol 1 pp 415ndash4182010

[32] Y L Krishnamurthy S B Naik and S Jayaram ldquoFungal com-munities in herbaceous medicinal plants from the malnadregion Southern IndiardquoMicrobes and Environments vol 23 no1 pp 24ndash28 2008

[33] D L Hawksworth and A Y Rossman ldquoWhere are all theundescribed fungirdquo Phytopathology vol 87 no 9 pp 888ndash8911997

[34] D Wilson and G C CarrolL ldquoInfection studies of Disculaquercina an endophyte of Quercus garryanardquo Mycologia vol86 no 5 pp 635ndash647 1994

[35] G A Strobel W M Hess J-Y Li et al ldquoPestalotiopsis guepiniia taxol-producing endophyte of the wollemi pine Wollemianobilisrdquo Australian Journal of Botany vol 45 no 6 pp 1073ndash1082 1997

[36] K Clay ldquoFungal endophytes of grassesrdquo Annual Review ofEcology and Systematics vol 21 no 1 pp 275ndash297 1990

[37] P Bayman L L Lebron R L Tremblay and D J Lodge ldquoVari-ation in endophytic fungi from roots and leaves of Lepanthes(Orchidaceae)rdquoNewPhytologist vol 135 no 1 pp 143ndash149 1997

[38] A EArnold ZMaynard andG SGilbert ldquoFungal endophytesin dicotyledonous neotropical trees patterns of abundance and

International Journal of Biodiversity 9

diversityrdquo Mycological Research vol 105 no 12 pp 1502ndash15072001

[39] P F Cannon andCM Simmons ldquoDiversity andhost preferenceof leaf endophytic fungi in the Iwokrama Forest ReserveGuyanardquoMycologia vol 94 no 2 pp 210ndash220 2002

[40] X Sun Q Ding K D Hyde and L D Guo ldquoCommunitystructure and preference of endophytic fungi of three woodyplants in a mixed forestrdquo Fungal Ecology vol 5 no 5 pp 624ndash632 2012

[41] P HollidayADictionary of Plant Pathology CambridgeUniver-sity Press Cambridge UK 1998

[42] J Collado G Platas and F Pelaez ldquoIdentification of an endo-phytic Nodulisporium sp from Quercus ilex in central Spainas the anamorph of Biscogniauxia mediterranea by rDNAsequence analysis and effect of different ecological factors ondistribution of the fungusrdquo Mycologia vol 93 no 5 pp 875ndash886 2001

[43] X-X Gao H Zhou D-Y Xu C-H Yu Y-Q Chen and L-HQu ldquoHigh diversity of endophytic fungi from the pharmaceuti-cal plant Heterosmilax japonica Kunth revealed by cultivation-independent approachrdquo FEMS Microbiology Letters vol 249no 2 pp 255ndash266 2005

[44] N S Raviraja ldquoFungal endophytes in five medicinal plantspecies from Kudremukh Range Western Ghats of IndiardquoJournal of Basic Microbiology vol 45 no 3 pp 230ndash235 2005

[45] A E Arnold Z Maynard G S Gilbert P D Coley and T AKursar ldquoAre tropical fungal endophytes hyperdiverserdquo EcologyLetters vol 3 no 4 pp 267ndash274 2000

[46] B S Naik J Shashikala and Y L Krishnamurthy ldquoDiversityof fungal endophytes in shrubby medicinal plants of Malnadregion Western Ghats Southern Indiardquo Fungal Ecology vol 1no 2-3 pp 89ndash93 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

International Journal of Biodiversity 3

0

20

40

60

80

100

Stem Root Rhizome InflPlant parts

MonsoonWinter

Relat

ive i

sola

tion

()

Figure 1 Relative seasonal isolations of fungal endophytes fromplant parts of medicinal species

The percentage of dominant endophytes (119863) was calculatedbased on the CF divided by the total number of endophytestimes 100 [20]

Differences in the extent of colonization of the sam-ples were analyzed by univariant analysis of variance (one-way ANOVA) and Tukeyrsquos honestly significant difference(HSD) as post hoc test using the statistical softwareSPSS160 The fungal isolations were considered for anal-ysis of ANOVA and Tukeyrsquos HSD Simpson and Shannondiversity indices were calculated for endophytic fungifrom different seasons with Estimate 119878 software (version6 httpviceroyeebuconneduestimates) Species richnesswas calculated using the onlineweb page rarefactor calculator(httpwww2biologyualbertacajbrzustorarefactphp)

Rarefaction indices were employed to compare thespecies richness among the plant species during two seasonsThe expected number of species in 119873 isolations was calcu-lated [21] Unweighted pair group method with arithmeticmean (UPGMA) cluster analysis was applied for all theisolates from plant species based on the number of isolatesrecovered from each plant species using a dendrogram con-structed based on Neirsquos genetic distances [22] using tools forpopulation genetics analysis (TFPGA) software [23]

3 Results

A total of 1529 isolates were obtained from 5200 tissuefragments from seven medicinal plant species The extentof endophytes colonization varied in plant parts with stemfragments harboring 80 of endophytic isolates followed byroot (1922) In other plant parts colonization was lowerIsolations of endophytes from various plant parts showedgreater numbers of endophytes during monsoon than winter(Figure 1) The high isolation rates (IR) of fungal endophyteswere recorded as 141 to 158 for T asthmatica in bothseasons while in Zingiber sp low rates of isolations

TA HA PHY RC EF PZ ZN

Rate

of i

sola

tion

Medicinal plants

MonsoonWinter

2

18

16

14

12

1

08

06

04

02

0

Figure 2 Seasonal isolation rates of fungal endophytes frommedic-inal species Endophytic fungi were isolated from seven medicinalplant species during monsoon and winter seasons respectively

were obtained (Figure 2) Thirty-one fungal taxa wereidentified which consisted of coelomycetes (65)hyphomycetes (32) and ascomycete isolations of 3The frequency of fungal colonization (CF) differedamong the seven plant species (Table 2) Fusarium spAcremonium Chaetomium and Phoma are some of theendophytes with high colonization frequency The dominantfungal genera include Fusarium spp (119863 = 1064) andAcremonium (119863 = 948) Few endophytic fungi such asA strictum had wide distributions in host plants and wereisolated from most plants with the exception of Zingibersp and Plumbago zeylanica whereas species of FusariumTrichoderma Curvularia and Penicillium were isolated frommore than three plant species

Host-specificity was observed for few of the fungalendophytes isolated from two of the seven medicinal plants(Table 2) Colletotrichum dematium Nigrospora oryzaeHeinesia rubi Pestalotiopsis guepinii and unidentified redpycnidial form were isolated from the stem segments of Tasthmatica only while in Rubia cordifolia one endophyticPericonia exhibited specificity P islandicum and T viridewere isolated from root segments of Phyllanthus amarus

Diversity indices of fungal endophytes variedwithin plantspecies as well as between seasons (Table 3) High Shannon-Weiner diversity index was recorded for T asthmatica (1198671 =260) and P amarus (1198671 = 227) during monsoon and winterseasons whereas low indices were recorded for E foetidumand Zingiber during monsoon and winter seasons respec-tively 42 of the total 31 taxa were found inmonsoon seasonwhile 55 of them colonized in both seasons Simpson index(1119897) was high for T asthmatica with a richness of 19 fungalspecies during monsoon season while P amarus recordedhighest richness of species during winter season Rarefac-tion curves calculated for the endophytic fungal isolationsindicated maximum species richness for T asthmatica and Pamarus during monsoon and winter isolations respectively

4 International Journal of Biodiversity

Table2Colon

izationfre

quencylowast

ofendo

phyticfung

iisolated

from

plantp

artsof

sevenmedicinalplantspecies

Season

sMon

soon

Winter

Plantspecies

TARC

PPH

YEF

CAZN

TARC

PZPH

YEF

CAZN

119863(

)PlantP

arts

Stem

SR

SR

ISt

RI

SRh

RStem

SR

SR

StR

IS

RhR

Endo

phytes

Acremonium

strictum

250

160

mdash100

9080

90mdash

20

90mdash

mdashmdash

mdash125

75150

mdash50

25

60

50

35

mdashmdash

mdashmdash

948

Alternariaalternata

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash85

mdashmdash

mdash15

mdashmdash

mdashmdash

mdashmdash

mdash065

Aspergillus

terreus

115

mdash50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash70

mdashmdash

mdashmdash

mdashmdash

mdashmdash

154

Redpycnidia

50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

033

Botryodiplodiatheobrom

ae25

75mdash

25

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash50

mdash25

mdashmdash

15mdash

mdashmdash

mdashmdash

mdash14

1Cladosporiu

mherbarum

mdashmdash

mdash25

mdashmdash

mdashmdash

mdashmdash

mdashmdash

05

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

020

Colletotrichum

dematium

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

200

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash13

1C

lindemuthianum

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash160

mdashmdash

mdashmdash

100

35

mdashmdash

mdashmdash

60

mdashmdash

mdashmdash

mdashmdash

232

Chaetomium

globosum

275

mdashmdash

mdashmdash

mdash160

mdashmdash

mdashmdash

mdash10

mdash160

mdash35

mdashmdash

50

mdashmdash

mdashmdash

mdashmdash

mdash451

Curvularialunata

mdashmdash

mdash25

mdashmdash

mdashmdash

60

40

mdashmdash

mdashmdash

mdash100

mdash75

mdashmdash

mdashmdash

25

mdashmdash

mdashmdash

212

Fusariu

msp

mdashmdash

mdashmdash

15mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

15mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

020

Fgram

inearum

mdashmdash

mdashmdash

mdashmdash

mdashmdash

95mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash10

mdashmdash

mdashmdash

mdash069

Foxysporum

75200

60

mdashmdash

mdashmdash

1035

mdashmdash

mdashmdash

05

mdash15

mdashmdash

mdashmdash

1040

mdashmdash

mdash10

mdash301

Fsolani

50

100

mdashmdash

45

mdash50

mdashmdash

100

mdashmdash

mdash40

mdashmdash

mdash45

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash281

Fpseudonygamai

mdashmdash

mdashmdash

mdash25

mdashmdash

mdashmdash

mdashmdash

mdash05

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash020

Fverticilloides

210

mdash55

100

mdash75

mdashmdash

mdashmdash

20

mdashmdash

mdashmdash

110

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash373

Gela

sinospora

spp

25

mdashmdash

20

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

029

Hein

esiarubi

15mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash010

Mem

noniellaspp

85

25

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash069

Myrotheciu

mverrucaria

30

mdash75

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

60

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash10

8Nigrospora

oryzae

50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

033

Peric

oniasp

mdash175

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash114

Pesta

lotio

psisguepinii

20

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

013

Pesta

lotio

psissp

mdashmdash

mdash50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash20

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash059

Phom

aspp

60

100

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

90mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash16

3Ph

omopsis

sp

50

mdashmdash

mdashmdash

mdashmdash

mdash10

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash039

Penicilliu

msp

100

45

mdashmdash

20

mdash10

mdashmdash

30

mdashmdash

mdashmdash

mdash20

50

25

mdashmdash

mdash15

mdashmdash

mdashmdash

206

Pislandicum

mdashmdash

mdash75

10mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

055

Trich

odermaharzianu

mmdash

mdash75

mdashmdash

45

30

mdashmdash

mdashmdash

mdash15

mdashmdash

mdashmdash

50

15mdash

mdashmdash

50

mdashmdash

20

mdash19

6Tvirid

emdash

mdashmdash

mdash50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

10mdash

mdashmdash

mdashmdash

mdashmdash

mdash039

Sphaeronem

asp

25

mdashmdash

120

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

082

Verticillium

albo-atru

mmdash

mdash35

mdashmdash

mdashmdash

mdashmdash

mdashmdash

10mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash029

Pycnidialforms

mdash75

150

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

147

Sterile

mycelia

60

mdash35

50

mdash10

mdashmdash

mdashmdash

mdashmdash

mdash20

mdashmdash

45

mdashmdash

mdashmdash

mdashmdash

mdashmdash

144

Total

314

191

107

126

4647

682

4479

06

610

143

7263

7334

1829

2025

00

60

1529

314

191

107

172

117

123

22143

7263

107

4745

61529

lowast

200segm

entswerep

lated

forfrequ

ency

analysisdu

ringmon

soon

andwinterseasonsT

ATylophora

asthmatica

RC

RubiacordifoliaP

ZPlum

bago

zeylanica

PHY

Phyllanthu

samarusE

FEryngium

foetidum

CA

CentellaasiaticaZN

ZingiberspSstemStsto

lon

RrootR

hrhizom

eIinflo

rescence

International Journal of Biodiversity 5

Table 3 Diversity indices (1198671) and species richness of the medicinal plants

Monsoon Winter

Plant species Total isolates Total species richness Diversity indices Total isolates Total species richness Diversity indicesSimpson Shannon Simpson Shannon

Tylophora asthmatica 314 19 090 260 143 70 078 166Rubia cordifolia 191 9 086 205 72 60 079 162Plumbago zeylanica 107 8 084 196 63 4 063 114Phyllanthus amarus 172 14 087 229 107 110 089 227Eryngium foetidum 117 9 077 168 47 6 075 153Centella asiatica 123 8 083 188 45 6 076 157Zingiber sp 22 8 079 181 6 2 044 064

20

18

16

14

12

10

8

6

4

2

01 25 50 74 95 120 140 165 190

Number of isolations

TARCPZPHY

EFCAZN

Monsoon

Expe

cted

num

ber o

f spe

cies

(E(s))

Figure 3 Rarefaction curvesof fungal endophytes from monsoonisolations (number of isolations versus expected number of speciesE(119904)

) TA Tylophora asthmatica RC Rubia cordifolia PZ Plumbagozeylanica PHY Phyllanthus amarus EF Eryngium foetidum CACentella asiatica and ZN Zingiber sp

(Figures 3 and 4) Differences in the number of isolates andcolonization frequency differed significantly between seasons(119875 lt 0001) as indicated in Table 4

Neirsquos genetic distance between endophytes isolated fromplant species ranged from 03185 (between populations ofZingiber and C asiatica) to 09116 (between populationsof Zingiber and T asthmatica) (Table 5) which was widelyranged This indicates a closer relationship between endo-phytic fungal patterns of both plants In general fungalspecies from T asthmatica is most distanced from the other

TARCPZPHY

EFCAZN

Winter

12

10

8

6

4

2

01 7 13 19 25 31 37 43 49 55

Number of isolations

Expe

cted

num

ber o

f spe

cies

(E(s))

Figure 4 Rarefaction curves of fungal endophytes from winterisolations (number of isolations versus expected number of speciesE(119904)

) TA Tylophora asthmatica RC Rubia cordifolia PZ Plumbagozeylanica PHY Phyllanthus amarus EF Eryngium foetidum CACentella asiatica and ZN Zingiber sp

plants studied In order to represent the relationships amongplant species cluster analysis (UPGMA)was used to generatea dendrogram based on Neirsquos genetic distances betweenpopulations (Figure 5) In this dendrogram all plants forma distinct cluster When the transect line was placed atapproximately 04 on the distance scale two distinct groupswere formed The first cluster was formed by CA-ZN while

6 International Journal of Biodiversity

Table 4 ANOVA table of seasonal variation of endophytic fungi analyzed from seven medicinal plant species

Sum of squares Degrees of freedom Mean2119865 Significance

Between seasons 119319 26 458919 34382 0001Within seasons 1001062 75 13346 mdash mdashTotal 1293296 101 mdash mdash mdash

Table 5 Neirsquos genetic distance of plant species analyzed for endo-phytic fungi

TA RC PZ PHY EF CA ZNTA lowastlowastlowast 07885 05008 06061 04055 07885 09116RC lowastlowastlowast 05008 07239 04055 03610 06061PZ lowastlowastlowast 06633 03610 05008 04520PHY lowastlowastlowast 04520 05008 06633EF lowastlowastlowast 04055 03610CA lowastlowastlowast 03185ZN lowastlowastlowast

lowastIndicates that genetic distance between the same genus does not exist it isshown for different genera

0700 0525 0350 0175 0000

CA

ZN

PZ

EF

RC

PHY

TA

Figure 5 UPGMA cluster analysis of fungal endophytes represent-ing seven medicinal plant species Plant codes are TA Tylophoraasthmatica RC Rubia cordifolia PZ Plumbago zeylanica PHYPhyllanthus amarus EF Eryngium foetidum CA Centella asiaticaand ZN Zingiber sp

PZ-EF formed the second cluster PHY RC and TA are foundoutside the cluster

4 Discussion

Medicinal plants are considered as a repository of ldquoendo-phytic microbesrdquo living in the internal tissues of plants Thequest for identifying novel bioactives from the endophyticfungi has resulted in the sampling of host plants such asherbs shrubs tree species and vines in unique places ofecological adaptations around the rainforests of the worldSuch niches harbor great species diversity unintervened by

human activities Efforts in this direction to sample plantslocated in the rainforests around the world with potentialethnomedicinal values have resulted in the isolation of fungalendophytes unique to a particular plant species with distinctbioactivity

41 Endophyte Colonization inMedicinal Species Themedic-inal plant species were sampled from the Talacauvery sub-cluster situated in the Kodagu District of Western Ghats ofSouthern India This region is among one of the 34 hot spotsof biodiversity Recently a proposal to include this biodiver-sity spot in the list of UNESCOHeritage cluster site is under-way (httpwwwatreeorgwg unesco whs) The natives aswell as the ethnic tribes inhabiting this region still dependon the plants as a source of medicine to cure some of theailments [12] Seven medicinal plant species assigned to sixplant families were selected for the study in natural popula-tions in two seasons from a single location from the studyarea stretching over an area of 25 kilometers Sampling wasconducted during monsoon and winter seasons as two ofthe herbaceous species E foetidum and Zingiber sp growonly till the second half of the year (June to December) andtheir nonavailability during summer (March toMay)makes itdifficult to consider the summer season for endophyticanalysis

From 5200 segments of plant materials a total of 1529isolates were obtained these were grouped into 31 taxaMycelia sterilia the fungal taxa that failed to sporulate werealso reported from this study This fungal group is prevalentin endophytic studies [24] The fungal endophytes wereanalyzed from four plant parts namely stem root rhizomeand inflorescence however their occurrence in root andinflorescence was investigated for few plant species only asthe phenology and sampling of plants never correlated withseasons The leaves were not considered for isolations sincesome of the plants were climbers and stragglers with delicatehairy surfaces and stringent surface sterilization techniqueswould render them unsuitable for plating on agar mediumRelative percentages of endophytic isolations from stemsegments were greater (8037) than isolations from roots(1922) Our results are supported by the earlier work ofHuang et al [25] on 29 traditional Chinese medicinal plantsthat fungal endophytes aremore frequent in stem tissues thanroots Among the fungal taxa coelomycete isolations weremore dominant than hyphomycetes and have been found inearlier studies in endophytes of tree species [26]

Endophytes such asColletotrichum PhomaAcremoniumChaetomium Botryodiplodia and Trichoderma were isolatedwith 119863 gt 20 Few fungal taxa that are less frequently iso-lated are Pestalotiopsis Penicillium islandicum Cladosporiumherbarum Alternaria alternata F graminearum Phomopsis

International Journal of Biodiversity 7

and Sphaeronema Colletotrichum spp are the most fre-quently encountered endophytic fungi [18] whereas Pestalo-tiopsis spp are well documented as endophytes of many rain-forest plants [27 28] tropical tree species namelyTerminaliaarjuna [29] Azadirachta indica [30] and many herbs andshrubs [25 31 32] It is necessary to screen newer plant speciesfor the isolation of fungal endophytes as Hawksworth andRossman [33] estimate that there are still millions of speciesof fungi yet to be identified Differences in the colonizationfrequencies of endophytes during two seasons were observedand more isolation during monsoon season is due to the factthat the slimy conidia of fungal spores are dispersed betterby rain splashes and germination of conidia is influenced byclimatic factors [34]

42 Host-Specificity of Fungal Endophytes We observed thatsome fungal taxa exhibited host-specificity a phenomenonoften associated with endophytes Three plant species Tasthmatica R cordifolia and P amarus were host-specificto endophytes The red pycnidial endophyte (TA-005) wasisolated from the stem fragments of T asthmatica onlysuggesting the host-specificity of this endophyte Pestalo-tiopsis guepinii was isolated from the stem segments of Tasthmatica It has been reported as an endophyte ofWollemianobilis growing in Sydney Australia [35] Heinesia rubiP islandicum and TA-005 are new reports of fungi asendophytes Host-specificity of endophytic fungi has beenobserved earlier for grasses [36] orchids [37] and forest treespecies [38 39] Recently Sun et al [40] reiterated the termldquohost-specificityrdquo as taxa that occur exclusively on a statedhost but not on other hosts in the same habitat [41] Ourstudies also indicate the host-specificity of endophytes as theplant species were sampled from a single habitat

43 Seasonal Diversity of Fungal Endophytes Diversityindices for fungal endophytes as analyzed by Shannon-Weiner (1198671) and Simpson (1119897) indices indicated differencesin seasonal variation and species richness High indices werenoted for T asthmatica (1198671 = 26) and P amarus (1198671 = 227)during monsoon and winter seasons respectivelyThe fungalspecies did not differ significantly between plant specieswhereas they differed between seasons (119875 lt 0001) Seasonalvariation in fungal isolates and colonization frequency hasbeen reported for many host plants [42 43] High coloniza-tion frequency as well as the species richness of endophyticfungi is limited to leaf segments rather than stem or bark seg-ments of host plants sampled from five medicinal species ofKudremukh Region of Western Ghats [44] Species richnessin our study is limited to stem fragments among the plantparts considered for analysis

Most studies on fungal endophytes in tropics haverevealed remarkable patterns of endophyte colonizationand estimates of diversity in foliages of forest tree speciesrepresenting various sites such as Panamanian Forest [45]and Iwokrama Forest Reserve Guyana [39] In the NilgiriBiosphere Reserve Western Ghats India 75 dicotyledonousspecies in three different tropical forest types were sampledto study foliar endophytes and diversity [10] The endophyte

diversity in forest types was limited due to loose hostaffiliations among endophytes Studies on foliar endophytesfrom the sampling of herbaceous and shrubby medicinalplant species have revealed differences in the colonizationrates as well as seasonal diversity inMalnad Region of BhadraWildlife Sanctuary in Southern India [32 46]

The present study provides firsthand information onthe diversity and seasonal influence on the colonizationfrequencies of endophytic fungi from selected medicinalplants from one of the subclusters of biodiversity hot spotsin the Western Ghats of Southern India Although theisolation and analysis of endophyte communities in herbsshrubs and trees are not uncommon each of the studies isunique with reference to number of hosts species of fungalendophytes and their specificityThe fungal endophytes havebeen subjected to fermentation studies and extracts are beingtested for biological activities

5 Conclusion

The study provides firsthand information on the diversityand seasonal influence on the colonization frequencies ofendophytic fungi from sevenmedicinal plants fromone of thesubclusters of biodiversity hot spots in the Western Ghats ofSouthern IndiaThe present investigation is the first isolationof endophytes from the medicinal species and their plantpartsThough isolation of endophytes has been accomplishedfrom various forest types and locations around the globeeach study is unique in documenting newer endophytic taxaWe are currently working on the fermentation of fungalendophytes to obtain newer antioxidants with therapeuticapplications

Conflict of Interests

The authors declare no conflict of interests regarding thepublication of this paper

Acknowledgments

This work was carried out with the financial assistancefrom the Department of Science amp Technology (DST)-SERCDivision Government of India under the Women Scien-tist Scheme [DST-WOS (A)] awarded to Monnanda Soma-iah Nalini (DST sanction no SRWOS (A)LS-762006 dt02082007) Monnanda Somaiah Nalini is thankful to theChairman DOS in Biotechnology for providing the neces-sary facilities for the completion of the project

References

[1] K Clay J Holah and J A Rudgers ldquoHerbivores cause a rapidincrease in hereditary symbiosis and alter plant communitycompositionrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 102 no 35 pp 12465ndash124702005

[2] P Alvarez-Loayza J F White Jr M S Torres et al ldquoLight con-verts endosymbiotic fungus to pathogen influencing seedling

8 International Journal of Biodiversity

survival and niche-space filling of a common tropical treeIriartea deltoideardquo PloS ONE vol 6 no 1 2011

[3] R Linnakoski H Puhakka-tarvainen andA Pappinen ldquoEndo-phytic fungi isolated from Khaya anthotheca in Ghanardquo FungalEcology vol 5 no 3 pp 298ndash308 2012

[4] J F White Jr and M S Torres ldquoIs plant endophyte-mediateddefensive mutualism the result of oxidative stress protectionrdquoPhysiologia Plantarum vol 138 no 4 pp 440ndash446 2010

[5] H-Y Li D-W Li C-M He Z-P Zhou T Mei and H-MXu ldquoDiversity and heavy metal tolerance of endophytic fungifrom six dominant plant species in a Pb-Zn mine wasteland inChinardquo Fungal Ecology vol 5 no 3 pp 309ndash315 2012

[6] A E Arnold ldquoUnderstanding the diversity of foliar endo-phytic fungi progress challenges and frontiersrdquo Fungal BiologyReviews vol 21 no 2-3 pp 51ndash66 2007

[7] K D Hyde and K Soytong ldquoThe fungal endophyte dilemmardquoFungal Diversity vol 33 pp 163ndash173 2008

[8] G Strobel and B Daisy ldquoBioprospecting for microbial endo-phytes and their natural productsrdquoMicrobiology and MolecularBiology Reviews vol 67 no 4 pp 491ndash502 2003

[9] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007

[10] T S Suryanarayanan T S Murali N Thirunavukkarasu M BGovinda Rajulu G Venkatesan and R Sukumar ldquoEndophyticfungal communities in woody perennials of three tropical foresttypes of the Western Ghats southern Indiardquo Biodiversity andConservation vol 20 no 5 pp 913ndash928 2011

[11] R A Mittermeier N Myers P R Gil and C G MittermeierHotspots Earthrsquos Biologically Richest and Most Endangered Ter-restrial Ecoregions Cemex Conservation International Wash-ington DC USA 2000

[12] R D Kshirsagar and N P Singh ldquoSome less known eth-nomedicinal uses from Mysore and Coorg districts Karnatakastate Indiardquo Journal of Ethnopharmacology vol 75 no 2-3 pp231ndash238 2001

[13] N YoganarasimhanMedicinal Plants of India Karnataka Inter-line Publishing Bangalore India 1996

[14] B Schulz U Wanke S Draeger and H J Aust ldquoEndophytesfrom herbaceous plants and shrubs effectiveness of surfacesterilization methodsrdquo Mycological Research vol 97 no 12 pp1447ndash1450 1993

[15] H Barnett and B Hunter Illustrated Genera of Imperfect FungiBurgess Publishing Minneapolis Minn USA 1998

[16] K H DomschW Gams and T AndersonCompendium of SoilFungi Academic Press New York NY USA 2003

[17] J F Leslie and B A Summerell The Fusarium LaboratoryManual Blackwell Publishing London UK 2006

[18] W Photita P W J Taylor R Ford K D Hyde and S Lumy-ong ldquoMorphological and molecular characterization of Col-letotrichum species from herbaceous plants inThailandrdquo FungalDiversity vol 18 pp 117ndash133 2005

[19] V Kumaresan and T S Suryanarayanan ldquoOccurrence and dis-tribution of endophytic fungi in a mangrove communityrdquoMycological Research vol 105 no 11 pp 1388ndash1391 2001

[20] V Kumaresan and T S Suryanarayanan ldquoEndophyte assem-blages in young mature and senescent leaves of Rhizophora

apiculata evidence for the role of endophytes inmangrove litterdegradationrdquo Fungal Diversity vol 9 pp 81ndash91 2002

[21] J A Ludwig and J F Reynolds Statistical Ecology A Primer onMethods and Computing John Wiley amp Sons New York NYUSA 1988

[22] M Nei ldquoGenetic distance between populationsrdquo AmericanNaturalist vol 106 no 949 pp 283ndash293 1972

[23] M P Miller Tools For Population Genetic Analyses (TFPGA)V L3 A Windows program For the Analysis of Allozyme AndmoleculAr Genetic data Department of Biological SciencesNorthern Arizona University Flagstaff Ariz USA 1997

[24] D C Lacap K D Hyde and E C Y Liew ldquoAn evaluationof the fungal rsquomorphotypersquo concept based on ribosomal DNAsequencesrdquo Fungal Diversity vol 12 pp 53ndash66 2003

[25] W Y Huang Y Z Cai K D Hyde H Corke and M SunldquoBiodiversity of endophytic fungi associated with 29 traditionalChinese medicinal plantsrdquo Fungal Diversity vol 33 pp 61ndash752008

[26] M V Tejesvi B Mahesh M S Nalini et al ldquoFungal endophyteassemblages from ethnopharmaceutically important medicinaltreesrdquo Canadian Journal of Microbiology vol 52 no 5 pp 427ndash435 2006

[27] N Raj Coelomycetous Anamorphs With Appendage BearingConidia Edwards Brothers Ann Harbor Michigan 1993

[28] G A Strobel ldquoMicrobial gifts from rain forestsrdquo CanadianJournal of Plant Pathology vol 24 no 1 pp 14ndash20 2002

[29] M V Tejesvi B Mahesh M S Nalini et al ldquoEndophytic fungalassemblages from inner bark and twig of Terminalia arjunaW amp A (Combretaceae)rdquo World Journal of Microbiology andBiotechnology vol 21 no 8-9 pp 1535ndash1540 2005

[30] B Mahesh M V Tejesvi M S Nalini et al ldquoEndophyticmycoflora of inner bark of Azadirachta indica A Jussrdquo CurrentScience vol 88 no 2 pp 218ndash219 2005

[31] K Rajagopal S Kalavathy S Kokila et al ldquoDiversity of fungalendophytes in few medicinal herbs of southern Indiardquo AsianJournal of Experimental Biological Sciences vol 1 pp 415ndash4182010

[32] Y L Krishnamurthy S B Naik and S Jayaram ldquoFungal com-munities in herbaceous medicinal plants from the malnadregion Southern IndiardquoMicrobes and Environments vol 23 no1 pp 24ndash28 2008

[33] D L Hawksworth and A Y Rossman ldquoWhere are all theundescribed fungirdquo Phytopathology vol 87 no 9 pp 888ndash8911997

[34] D Wilson and G C CarrolL ldquoInfection studies of Disculaquercina an endophyte of Quercus garryanardquo Mycologia vol86 no 5 pp 635ndash647 1994

[35] G A Strobel W M Hess J-Y Li et al ldquoPestalotiopsis guepiniia taxol-producing endophyte of the wollemi pine Wollemianobilisrdquo Australian Journal of Botany vol 45 no 6 pp 1073ndash1082 1997

[36] K Clay ldquoFungal endophytes of grassesrdquo Annual Review ofEcology and Systematics vol 21 no 1 pp 275ndash297 1990

[37] P Bayman L L Lebron R L Tremblay and D J Lodge ldquoVari-ation in endophytic fungi from roots and leaves of Lepanthes(Orchidaceae)rdquoNewPhytologist vol 135 no 1 pp 143ndash149 1997

[38] A EArnold ZMaynard andG SGilbert ldquoFungal endophytesin dicotyledonous neotropical trees patterns of abundance and

International Journal of Biodiversity 9

diversityrdquo Mycological Research vol 105 no 12 pp 1502ndash15072001

[39] P F Cannon andCM Simmons ldquoDiversity andhost preferenceof leaf endophytic fungi in the Iwokrama Forest ReserveGuyanardquoMycologia vol 94 no 2 pp 210ndash220 2002

[40] X Sun Q Ding K D Hyde and L D Guo ldquoCommunitystructure and preference of endophytic fungi of three woodyplants in a mixed forestrdquo Fungal Ecology vol 5 no 5 pp 624ndash632 2012

[41] P HollidayADictionary of Plant Pathology CambridgeUniver-sity Press Cambridge UK 1998

[42] J Collado G Platas and F Pelaez ldquoIdentification of an endo-phytic Nodulisporium sp from Quercus ilex in central Spainas the anamorph of Biscogniauxia mediterranea by rDNAsequence analysis and effect of different ecological factors ondistribution of the fungusrdquo Mycologia vol 93 no 5 pp 875ndash886 2001

[43] X-X Gao H Zhou D-Y Xu C-H Yu Y-Q Chen and L-HQu ldquoHigh diversity of endophytic fungi from the pharmaceuti-cal plant Heterosmilax japonica Kunth revealed by cultivation-independent approachrdquo FEMS Microbiology Letters vol 249no 2 pp 255ndash266 2005

[44] N S Raviraja ldquoFungal endophytes in five medicinal plantspecies from Kudremukh Range Western Ghats of IndiardquoJournal of Basic Microbiology vol 45 no 3 pp 230ndash235 2005

[45] A E Arnold Z Maynard G S Gilbert P D Coley and T AKursar ldquoAre tropical fungal endophytes hyperdiverserdquo EcologyLetters vol 3 no 4 pp 267ndash274 2000

[46] B S Naik J Shashikala and Y L Krishnamurthy ldquoDiversityof fungal endophytes in shrubby medicinal plants of Malnadregion Western Ghats Southern Indiardquo Fungal Ecology vol 1no 2-3 pp 89ndash93 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

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BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

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Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

4 International Journal of Biodiversity

Table2Colon

izationfre

quencylowast

ofendo

phyticfung

iisolated

from

plantp

artsof

sevenmedicinalplantspecies

Season

sMon

soon

Winter

Plantspecies

TARC

PPH

YEF

CAZN

TARC

PZPH

YEF

CAZN

119863(

)PlantP

arts

Stem

SR

SR

ISt

RI

SRh

RStem

SR

SR

StR

IS

RhR

Endo

phytes

Acremonium

strictum

250

160

mdash100

9080

90mdash

20

90mdash

mdashmdash

mdash125

75150

mdash50

25

60

50

35

mdashmdash

mdashmdash

948

Alternariaalternata

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash85

mdashmdash

mdash15

mdashmdash

mdashmdash

mdashmdash

mdash065

Aspergillus

terreus

115

mdash50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash70

mdashmdash

mdashmdash

mdashmdash

mdashmdash

154

Redpycnidia

50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

033

Botryodiplodiatheobrom

ae25

75mdash

25

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash50

mdash25

mdashmdash

15mdash

mdashmdash

mdashmdash

mdash14

1Cladosporiu

mherbarum

mdashmdash

mdash25

mdashmdash

mdashmdash

mdashmdash

mdashmdash

05

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

020

Colletotrichum

dematium

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

200

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash13

1C

lindemuthianum

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash160

mdashmdash

mdashmdash

100

35

mdashmdash

mdashmdash

60

mdashmdash

mdashmdash

mdashmdash

232

Chaetomium

globosum

275

mdashmdash

mdashmdash

mdash160

mdashmdash

mdashmdash

mdash10

mdash160

mdash35

mdashmdash

50

mdashmdash

mdashmdash

mdashmdash

mdash451

Curvularialunata

mdashmdash

mdash25

mdashmdash

mdashmdash

60

40

mdashmdash

mdashmdash

mdash100

mdash75

mdashmdash

mdashmdash

25

mdashmdash

mdashmdash

212

Fusariu

msp

mdashmdash

mdashmdash

15mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

15mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

020

Fgram

inearum

mdashmdash

mdashmdash

mdashmdash

mdashmdash

95mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash10

mdashmdash

mdashmdash

mdash069

Foxysporum

75200

60

mdashmdash

mdashmdash

1035

mdashmdash

mdashmdash

05

mdash15

mdashmdash

mdashmdash

1040

mdashmdash

mdash10

mdash301

Fsolani

50

100

mdashmdash

45

mdash50

mdashmdash

100

mdashmdash

mdash40

mdashmdash

mdash45

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash281

Fpseudonygamai

mdashmdash

mdashmdash

mdash25

mdashmdash

mdashmdash

mdashmdash

mdash05

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash020

Fverticilloides

210

mdash55

100

mdash75

mdashmdash

mdashmdash

20

mdashmdash

mdashmdash

110

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash373

Gela

sinospora

spp

25

mdashmdash

20

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

029

Hein

esiarubi

15mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash010

Mem

noniellaspp

85

25

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash069

Myrotheciu

mverrucaria

30

mdash75

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

60

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash10

8Nigrospora

oryzae

50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

033

Peric

oniasp

mdash175

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash114

Pesta

lotio

psisguepinii

20

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

013

Pesta

lotio

psissp

mdashmdash

mdash50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash20

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash059

Phom

aspp

60

100

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

90mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash16

3Ph

omopsis

sp

50

mdashmdash

mdashmdash

mdashmdash

mdash10

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash039

Penicilliu

msp

100

45

mdashmdash

20

mdash10

mdashmdash

30

mdashmdash

mdashmdash

mdash20

50

25

mdashmdash

mdash15

mdashmdash

mdashmdash

206

Pislandicum

mdashmdash

mdash75

10mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

055

Trich

odermaharzianu

mmdash

mdash75

mdashmdash

45

30

mdashmdash

mdashmdash

mdash15

mdashmdash

mdashmdash

50

15mdash

mdashmdash

50

mdashmdash

20

mdash19

6Tvirid

emdash

mdashmdash

mdash50

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

10mdash

mdashmdash

mdashmdash

mdashmdash

mdash039

Sphaeronem

asp

25

mdashmdash

120

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

082

Verticillium

albo-atru

mmdash

mdash35

mdashmdash

mdashmdash

mdashmdash

mdashmdash

10mdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdash029

Pycnidialforms

mdash75

150

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

mdashmdash

147

Sterile

mycelia

60

mdash35

50

mdash10

mdashmdash

mdashmdash

mdashmdash

mdash20

mdashmdash

45

mdashmdash

mdashmdash

mdashmdash

mdashmdash

144

Total

314

191

107

126

4647

682

4479

06

610

143

7263

7334

1829

2025

00

60

1529

314

191

107

172

117

123

22143

7263

107

4745

61529

lowast

200segm

entswerep

lated

forfrequ

ency

analysisdu

ringmon

soon

andwinterseasonsT

ATylophora

asthmatica

RC

RubiacordifoliaP

ZPlum

bago

zeylanica

PHY

Phyllanthu

samarusE

FEryngium

foetidum

CA

CentellaasiaticaZN

ZingiberspSstemStsto

lon

RrootR

hrhizom

eIinflo

rescence

International Journal of Biodiversity 5

Table 3 Diversity indices (1198671) and species richness of the medicinal plants

Monsoon Winter

Plant species Total isolates Total species richness Diversity indices Total isolates Total species richness Diversity indicesSimpson Shannon Simpson Shannon

Tylophora asthmatica 314 19 090 260 143 70 078 166Rubia cordifolia 191 9 086 205 72 60 079 162Plumbago zeylanica 107 8 084 196 63 4 063 114Phyllanthus amarus 172 14 087 229 107 110 089 227Eryngium foetidum 117 9 077 168 47 6 075 153Centella asiatica 123 8 083 188 45 6 076 157Zingiber sp 22 8 079 181 6 2 044 064

20

18

16

14

12

10

8

6

4

2

01 25 50 74 95 120 140 165 190

Number of isolations

TARCPZPHY

EFCAZN

Monsoon

Expe

cted

num

ber o

f spe

cies

(E(s))

Figure 3 Rarefaction curvesof fungal endophytes from monsoonisolations (number of isolations versus expected number of speciesE(119904)

) TA Tylophora asthmatica RC Rubia cordifolia PZ Plumbagozeylanica PHY Phyllanthus amarus EF Eryngium foetidum CACentella asiatica and ZN Zingiber sp

(Figures 3 and 4) Differences in the number of isolates andcolonization frequency differed significantly between seasons(119875 lt 0001) as indicated in Table 4

Neirsquos genetic distance between endophytes isolated fromplant species ranged from 03185 (between populations ofZingiber and C asiatica) to 09116 (between populationsof Zingiber and T asthmatica) (Table 5) which was widelyranged This indicates a closer relationship between endo-phytic fungal patterns of both plants In general fungalspecies from T asthmatica is most distanced from the other

TARCPZPHY

EFCAZN

Winter

12

10

8

6

4

2

01 7 13 19 25 31 37 43 49 55

Number of isolations

Expe

cted

num

ber o

f spe

cies

(E(s))

Figure 4 Rarefaction curves of fungal endophytes from winterisolations (number of isolations versus expected number of speciesE(119904)

) TA Tylophora asthmatica RC Rubia cordifolia PZ Plumbagozeylanica PHY Phyllanthus amarus EF Eryngium foetidum CACentella asiatica and ZN Zingiber sp

plants studied In order to represent the relationships amongplant species cluster analysis (UPGMA)was used to generatea dendrogram based on Neirsquos genetic distances betweenpopulations (Figure 5) In this dendrogram all plants forma distinct cluster When the transect line was placed atapproximately 04 on the distance scale two distinct groupswere formed The first cluster was formed by CA-ZN while

6 International Journal of Biodiversity

Table 4 ANOVA table of seasonal variation of endophytic fungi analyzed from seven medicinal plant species

Sum of squares Degrees of freedom Mean2119865 Significance

Between seasons 119319 26 458919 34382 0001Within seasons 1001062 75 13346 mdash mdashTotal 1293296 101 mdash mdash mdash

Table 5 Neirsquos genetic distance of plant species analyzed for endo-phytic fungi

TA RC PZ PHY EF CA ZNTA lowastlowastlowast 07885 05008 06061 04055 07885 09116RC lowastlowastlowast 05008 07239 04055 03610 06061PZ lowastlowastlowast 06633 03610 05008 04520PHY lowastlowastlowast 04520 05008 06633EF lowastlowastlowast 04055 03610CA lowastlowastlowast 03185ZN lowastlowastlowast

lowastIndicates that genetic distance between the same genus does not exist it isshown for different genera

0700 0525 0350 0175 0000

CA

ZN

PZ

EF

RC

PHY

TA

Figure 5 UPGMA cluster analysis of fungal endophytes represent-ing seven medicinal plant species Plant codes are TA Tylophoraasthmatica RC Rubia cordifolia PZ Plumbago zeylanica PHYPhyllanthus amarus EF Eryngium foetidum CA Centella asiaticaand ZN Zingiber sp

PZ-EF formed the second cluster PHY RC and TA are foundoutside the cluster

4 Discussion

Medicinal plants are considered as a repository of ldquoendo-phytic microbesrdquo living in the internal tissues of plants Thequest for identifying novel bioactives from the endophyticfungi has resulted in the sampling of host plants such asherbs shrubs tree species and vines in unique places ofecological adaptations around the rainforests of the worldSuch niches harbor great species diversity unintervened by

human activities Efforts in this direction to sample plantslocated in the rainforests around the world with potentialethnomedicinal values have resulted in the isolation of fungalendophytes unique to a particular plant species with distinctbioactivity

41 Endophyte Colonization inMedicinal Species Themedic-inal plant species were sampled from the Talacauvery sub-cluster situated in the Kodagu District of Western Ghats ofSouthern India This region is among one of the 34 hot spotsof biodiversity Recently a proposal to include this biodiver-sity spot in the list of UNESCOHeritage cluster site is under-way (httpwwwatreeorgwg unesco whs) The natives aswell as the ethnic tribes inhabiting this region still dependon the plants as a source of medicine to cure some of theailments [12] Seven medicinal plant species assigned to sixplant families were selected for the study in natural popula-tions in two seasons from a single location from the studyarea stretching over an area of 25 kilometers Sampling wasconducted during monsoon and winter seasons as two ofthe herbaceous species E foetidum and Zingiber sp growonly till the second half of the year (June to December) andtheir nonavailability during summer (March toMay)makes itdifficult to consider the summer season for endophyticanalysis

From 5200 segments of plant materials a total of 1529isolates were obtained these were grouped into 31 taxaMycelia sterilia the fungal taxa that failed to sporulate werealso reported from this study This fungal group is prevalentin endophytic studies [24] The fungal endophytes wereanalyzed from four plant parts namely stem root rhizomeand inflorescence however their occurrence in root andinflorescence was investigated for few plant species only asthe phenology and sampling of plants never correlated withseasons The leaves were not considered for isolations sincesome of the plants were climbers and stragglers with delicatehairy surfaces and stringent surface sterilization techniqueswould render them unsuitable for plating on agar mediumRelative percentages of endophytic isolations from stemsegments were greater (8037) than isolations from roots(1922) Our results are supported by the earlier work ofHuang et al [25] on 29 traditional Chinese medicinal plantsthat fungal endophytes aremore frequent in stem tissues thanroots Among the fungal taxa coelomycete isolations weremore dominant than hyphomycetes and have been found inearlier studies in endophytes of tree species [26]

Endophytes such asColletotrichum PhomaAcremoniumChaetomium Botryodiplodia and Trichoderma were isolatedwith 119863 gt 20 Few fungal taxa that are less frequently iso-lated are Pestalotiopsis Penicillium islandicum Cladosporiumherbarum Alternaria alternata F graminearum Phomopsis

International Journal of Biodiversity 7

and Sphaeronema Colletotrichum spp are the most fre-quently encountered endophytic fungi [18] whereas Pestalo-tiopsis spp are well documented as endophytes of many rain-forest plants [27 28] tropical tree species namelyTerminaliaarjuna [29] Azadirachta indica [30] and many herbs andshrubs [25 31 32] It is necessary to screen newer plant speciesfor the isolation of fungal endophytes as Hawksworth andRossman [33] estimate that there are still millions of speciesof fungi yet to be identified Differences in the colonizationfrequencies of endophytes during two seasons were observedand more isolation during monsoon season is due to the factthat the slimy conidia of fungal spores are dispersed betterby rain splashes and germination of conidia is influenced byclimatic factors [34]

42 Host-Specificity of Fungal Endophytes We observed thatsome fungal taxa exhibited host-specificity a phenomenonoften associated with endophytes Three plant species Tasthmatica R cordifolia and P amarus were host-specificto endophytes The red pycnidial endophyte (TA-005) wasisolated from the stem fragments of T asthmatica onlysuggesting the host-specificity of this endophyte Pestalo-tiopsis guepinii was isolated from the stem segments of Tasthmatica It has been reported as an endophyte ofWollemianobilis growing in Sydney Australia [35] Heinesia rubiP islandicum and TA-005 are new reports of fungi asendophytes Host-specificity of endophytic fungi has beenobserved earlier for grasses [36] orchids [37] and forest treespecies [38 39] Recently Sun et al [40] reiterated the termldquohost-specificityrdquo as taxa that occur exclusively on a statedhost but not on other hosts in the same habitat [41] Ourstudies also indicate the host-specificity of endophytes as theplant species were sampled from a single habitat

43 Seasonal Diversity of Fungal Endophytes Diversityindices for fungal endophytes as analyzed by Shannon-Weiner (1198671) and Simpson (1119897) indices indicated differencesin seasonal variation and species richness High indices werenoted for T asthmatica (1198671 = 26) and P amarus (1198671 = 227)during monsoon and winter seasons respectivelyThe fungalspecies did not differ significantly between plant specieswhereas they differed between seasons (119875 lt 0001) Seasonalvariation in fungal isolates and colonization frequency hasbeen reported for many host plants [42 43] High coloniza-tion frequency as well as the species richness of endophyticfungi is limited to leaf segments rather than stem or bark seg-ments of host plants sampled from five medicinal species ofKudremukh Region of Western Ghats [44] Species richnessin our study is limited to stem fragments among the plantparts considered for analysis

Most studies on fungal endophytes in tropics haverevealed remarkable patterns of endophyte colonizationand estimates of diversity in foliages of forest tree speciesrepresenting various sites such as Panamanian Forest [45]and Iwokrama Forest Reserve Guyana [39] In the NilgiriBiosphere Reserve Western Ghats India 75 dicotyledonousspecies in three different tropical forest types were sampledto study foliar endophytes and diversity [10] The endophyte

diversity in forest types was limited due to loose hostaffiliations among endophytes Studies on foliar endophytesfrom the sampling of herbaceous and shrubby medicinalplant species have revealed differences in the colonizationrates as well as seasonal diversity inMalnad Region of BhadraWildlife Sanctuary in Southern India [32 46]

The present study provides firsthand information onthe diversity and seasonal influence on the colonizationfrequencies of endophytic fungi from selected medicinalplants from one of the subclusters of biodiversity hot spotsin the Western Ghats of Southern India Although theisolation and analysis of endophyte communities in herbsshrubs and trees are not uncommon each of the studies isunique with reference to number of hosts species of fungalendophytes and their specificityThe fungal endophytes havebeen subjected to fermentation studies and extracts are beingtested for biological activities

5 Conclusion

The study provides firsthand information on the diversityand seasonal influence on the colonization frequencies ofendophytic fungi from sevenmedicinal plants fromone of thesubclusters of biodiversity hot spots in the Western Ghats ofSouthern IndiaThe present investigation is the first isolationof endophytes from the medicinal species and their plantpartsThough isolation of endophytes has been accomplishedfrom various forest types and locations around the globeeach study is unique in documenting newer endophytic taxaWe are currently working on the fermentation of fungalendophytes to obtain newer antioxidants with therapeuticapplications

Conflict of Interests

The authors declare no conflict of interests regarding thepublication of this paper

Acknowledgments

This work was carried out with the financial assistancefrom the Department of Science amp Technology (DST)-SERCDivision Government of India under the Women Scien-tist Scheme [DST-WOS (A)] awarded to Monnanda Soma-iah Nalini (DST sanction no SRWOS (A)LS-762006 dt02082007) Monnanda Somaiah Nalini is thankful to theChairman DOS in Biotechnology for providing the neces-sary facilities for the completion of the project

References

[1] K Clay J Holah and J A Rudgers ldquoHerbivores cause a rapidincrease in hereditary symbiosis and alter plant communitycompositionrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 102 no 35 pp 12465ndash124702005

[2] P Alvarez-Loayza J F White Jr M S Torres et al ldquoLight con-verts endosymbiotic fungus to pathogen influencing seedling

8 International Journal of Biodiversity

survival and niche-space filling of a common tropical treeIriartea deltoideardquo PloS ONE vol 6 no 1 2011

[3] R Linnakoski H Puhakka-tarvainen andA Pappinen ldquoEndo-phytic fungi isolated from Khaya anthotheca in Ghanardquo FungalEcology vol 5 no 3 pp 298ndash308 2012

[4] J F White Jr and M S Torres ldquoIs plant endophyte-mediateddefensive mutualism the result of oxidative stress protectionrdquoPhysiologia Plantarum vol 138 no 4 pp 440ndash446 2010

[5] H-Y Li D-W Li C-M He Z-P Zhou T Mei and H-MXu ldquoDiversity and heavy metal tolerance of endophytic fungifrom six dominant plant species in a Pb-Zn mine wasteland inChinardquo Fungal Ecology vol 5 no 3 pp 309ndash315 2012

[6] A E Arnold ldquoUnderstanding the diversity of foliar endo-phytic fungi progress challenges and frontiersrdquo Fungal BiologyReviews vol 21 no 2-3 pp 51ndash66 2007

[7] K D Hyde and K Soytong ldquoThe fungal endophyte dilemmardquoFungal Diversity vol 33 pp 163ndash173 2008

[8] G Strobel and B Daisy ldquoBioprospecting for microbial endo-phytes and their natural productsrdquoMicrobiology and MolecularBiology Reviews vol 67 no 4 pp 491ndash502 2003

[9] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007

[10] T S Suryanarayanan T S Murali N Thirunavukkarasu M BGovinda Rajulu G Venkatesan and R Sukumar ldquoEndophyticfungal communities in woody perennials of three tropical foresttypes of the Western Ghats southern Indiardquo Biodiversity andConservation vol 20 no 5 pp 913ndash928 2011

[11] R A Mittermeier N Myers P R Gil and C G MittermeierHotspots Earthrsquos Biologically Richest and Most Endangered Ter-restrial Ecoregions Cemex Conservation International Wash-ington DC USA 2000

[12] R D Kshirsagar and N P Singh ldquoSome less known eth-nomedicinal uses from Mysore and Coorg districts Karnatakastate Indiardquo Journal of Ethnopharmacology vol 75 no 2-3 pp231ndash238 2001

[13] N YoganarasimhanMedicinal Plants of India Karnataka Inter-line Publishing Bangalore India 1996

[14] B Schulz U Wanke S Draeger and H J Aust ldquoEndophytesfrom herbaceous plants and shrubs effectiveness of surfacesterilization methodsrdquo Mycological Research vol 97 no 12 pp1447ndash1450 1993

[15] H Barnett and B Hunter Illustrated Genera of Imperfect FungiBurgess Publishing Minneapolis Minn USA 1998

[16] K H DomschW Gams and T AndersonCompendium of SoilFungi Academic Press New York NY USA 2003

[17] J F Leslie and B A Summerell The Fusarium LaboratoryManual Blackwell Publishing London UK 2006

[18] W Photita P W J Taylor R Ford K D Hyde and S Lumy-ong ldquoMorphological and molecular characterization of Col-letotrichum species from herbaceous plants inThailandrdquo FungalDiversity vol 18 pp 117ndash133 2005

[19] V Kumaresan and T S Suryanarayanan ldquoOccurrence and dis-tribution of endophytic fungi in a mangrove communityrdquoMycological Research vol 105 no 11 pp 1388ndash1391 2001

[20] V Kumaresan and T S Suryanarayanan ldquoEndophyte assem-blages in young mature and senescent leaves of Rhizophora

apiculata evidence for the role of endophytes inmangrove litterdegradationrdquo Fungal Diversity vol 9 pp 81ndash91 2002

[21] J A Ludwig and J F Reynolds Statistical Ecology A Primer onMethods and Computing John Wiley amp Sons New York NYUSA 1988

[22] M Nei ldquoGenetic distance between populationsrdquo AmericanNaturalist vol 106 no 949 pp 283ndash293 1972

[23] M P Miller Tools For Population Genetic Analyses (TFPGA)V L3 A Windows program For the Analysis of Allozyme AndmoleculAr Genetic data Department of Biological SciencesNorthern Arizona University Flagstaff Ariz USA 1997

[24] D C Lacap K D Hyde and E C Y Liew ldquoAn evaluationof the fungal rsquomorphotypersquo concept based on ribosomal DNAsequencesrdquo Fungal Diversity vol 12 pp 53ndash66 2003

[25] W Y Huang Y Z Cai K D Hyde H Corke and M SunldquoBiodiversity of endophytic fungi associated with 29 traditionalChinese medicinal plantsrdquo Fungal Diversity vol 33 pp 61ndash752008

[26] M V Tejesvi B Mahesh M S Nalini et al ldquoFungal endophyteassemblages from ethnopharmaceutically important medicinaltreesrdquo Canadian Journal of Microbiology vol 52 no 5 pp 427ndash435 2006

[27] N Raj Coelomycetous Anamorphs With Appendage BearingConidia Edwards Brothers Ann Harbor Michigan 1993

[28] G A Strobel ldquoMicrobial gifts from rain forestsrdquo CanadianJournal of Plant Pathology vol 24 no 1 pp 14ndash20 2002

[29] M V Tejesvi B Mahesh M S Nalini et al ldquoEndophytic fungalassemblages from inner bark and twig of Terminalia arjunaW amp A (Combretaceae)rdquo World Journal of Microbiology andBiotechnology vol 21 no 8-9 pp 1535ndash1540 2005

[30] B Mahesh M V Tejesvi M S Nalini et al ldquoEndophyticmycoflora of inner bark of Azadirachta indica A Jussrdquo CurrentScience vol 88 no 2 pp 218ndash219 2005

[31] K Rajagopal S Kalavathy S Kokila et al ldquoDiversity of fungalendophytes in few medicinal herbs of southern Indiardquo AsianJournal of Experimental Biological Sciences vol 1 pp 415ndash4182010

[32] Y L Krishnamurthy S B Naik and S Jayaram ldquoFungal com-munities in herbaceous medicinal plants from the malnadregion Southern IndiardquoMicrobes and Environments vol 23 no1 pp 24ndash28 2008

[33] D L Hawksworth and A Y Rossman ldquoWhere are all theundescribed fungirdquo Phytopathology vol 87 no 9 pp 888ndash8911997

[34] D Wilson and G C CarrolL ldquoInfection studies of Disculaquercina an endophyte of Quercus garryanardquo Mycologia vol86 no 5 pp 635ndash647 1994

[35] G A Strobel W M Hess J-Y Li et al ldquoPestalotiopsis guepiniia taxol-producing endophyte of the wollemi pine Wollemianobilisrdquo Australian Journal of Botany vol 45 no 6 pp 1073ndash1082 1997

[36] K Clay ldquoFungal endophytes of grassesrdquo Annual Review ofEcology and Systematics vol 21 no 1 pp 275ndash297 1990

[37] P Bayman L L Lebron R L Tremblay and D J Lodge ldquoVari-ation in endophytic fungi from roots and leaves of Lepanthes(Orchidaceae)rdquoNewPhytologist vol 135 no 1 pp 143ndash149 1997

[38] A EArnold ZMaynard andG SGilbert ldquoFungal endophytesin dicotyledonous neotropical trees patterns of abundance and

International Journal of Biodiversity 9

diversityrdquo Mycological Research vol 105 no 12 pp 1502ndash15072001

[39] P F Cannon andCM Simmons ldquoDiversity andhost preferenceof leaf endophytic fungi in the Iwokrama Forest ReserveGuyanardquoMycologia vol 94 no 2 pp 210ndash220 2002

[40] X Sun Q Ding K D Hyde and L D Guo ldquoCommunitystructure and preference of endophytic fungi of three woodyplants in a mixed forestrdquo Fungal Ecology vol 5 no 5 pp 624ndash632 2012

[41] P HollidayADictionary of Plant Pathology CambridgeUniver-sity Press Cambridge UK 1998

[42] J Collado G Platas and F Pelaez ldquoIdentification of an endo-phytic Nodulisporium sp from Quercus ilex in central Spainas the anamorph of Biscogniauxia mediterranea by rDNAsequence analysis and effect of different ecological factors ondistribution of the fungusrdquo Mycologia vol 93 no 5 pp 875ndash886 2001

[43] X-X Gao H Zhou D-Y Xu C-H Yu Y-Q Chen and L-HQu ldquoHigh diversity of endophytic fungi from the pharmaceuti-cal plant Heterosmilax japonica Kunth revealed by cultivation-independent approachrdquo FEMS Microbiology Letters vol 249no 2 pp 255ndash266 2005

[44] N S Raviraja ldquoFungal endophytes in five medicinal plantspecies from Kudremukh Range Western Ghats of IndiardquoJournal of Basic Microbiology vol 45 no 3 pp 230ndash235 2005

[45] A E Arnold Z Maynard G S Gilbert P D Coley and T AKursar ldquoAre tropical fungal endophytes hyperdiverserdquo EcologyLetters vol 3 no 4 pp 267ndash274 2000

[46] B S Naik J Shashikala and Y L Krishnamurthy ldquoDiversityof fungal endophytes in shrubby medicinal plants of Malnadregion Western Ghats Southern Indiardquo Fungal Ecology vol 1no 2-3 pp 89ndash93 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

International Journal of Biodiversity 5

Table 3 Diversity indices (1198671) and species richness of the medicinal plants

Monsoon Winter

Plant species Total isolates Total species richness Diversity indices Total isolates Total species richness Diversity indicesSimpson Shannon Simpson Shannon

Tylophora asthmatica 314 19 090 260 143 70 078 166Rubia cordifolia 191 9 086 205 72 60 079 162Plumbago zeylanica 107 8 084 196 63 4 063 114Phyllanthus amarus 172 14 087 229 107 110 089 227Eryngium foetidum 117 9 077 168 47 6 075 153Centella asiatica 123 8 083 188 45 6 076 157Zingiber sp 22 8 079 181 6 2 044 064

20

18

16

14

12

10

8

6

4

2

01 25 50 74 95 120 140 165 190

Number of isolations

TARCPZPHY

EFCAZN

Monsoon

Expe

cted

num

ber o

f spe

cies

(E(s))

Figure 3 Rarefaction curvesof fungal endophytes from monsoonisolations (number of isolations versus expected number of speciesE(119904)

) TA Tylophora asthmatica RC Rubia cordifolia PZ Plumbagozeylanica PHY Phyllanthus amarus EF Eryngium foetidum CACentella asiatica and ZN Zingiber sp

(Figures 3 and 4) Differences in the number of isolates andcolonization frequency differed significantly between seasons(119875 lt 0001) as indicated in Table 4

Neirsquos genetic distance between endophytes isolated fromplant species ranged from 03185 (between populations ofZingiber and C asiatica) to 09116 (between populationsof Zingiber and T asthmatica) (Table 5) which was widelyranged This indicates a closer relationship between endo-phytic fungal patterns of both plants In general fungalspecies from T asthmatica is most distanced from the other

TARCPZPHY

EFCAZN

Winter

12

10

8

6

4

2

01 7 13 19 25 31 37 43 49 55

Number of isolations

Expe

cted

num

ber o

f spe

cies

(E(s))

Figure 4 Rarefaction curves of fungal endophytes from winterisolations (number of isolations versus expected number of speciesE(119904)

) TA Tylophora asthmatica RC Rubia cordifolia PZ Plumbagozeylanica PHY Phyllanthus amarus EF Eryngium foetidum CACentella asiatica and ZN Zingiber sp

plants studied In order to represent the relationships amongplant species cluster analysis (UPGMA)was used to generatea dendrogram based on Neirsquos genetic distances betweenpopulations (Figure 5) In this dendrogram all plants forma distinct cluster When the transect line was placed atapproximately 04 on the distance scale two distinct groupswere formed The first cluster was formed by CA-ZN while

6 International Journal of Biodiversity

Table 4 ANOVA table of seasonal variation of endophytic fungi analyzed from seven medicinal plant species

Sum of squares Degrees of freedom Mean2119865 Significance

Between seasons 119319 26 458919 34382 0001Within seasons 1001062 75 13346 mdash mdashTotal 1293296 101 mdash mdash mdash

Table 5 Neirsquos genetic distance of plant species analyzed for endo-phytic fungi

TA RC PZ PHY EF CA ZNTA lowastlowastlowast 07885 05008 06061 04055 07885 09116RC lowastlowastlowast 05008 07239 04055 03610 06061PZ lowastlowastlowast 06633 03610 05008 04520PHY lowastlowastlowast 04520 05008 06633EF lowastlowastlowast 04055 03610CA lowastlowastlowast 03185ZN lowastlowastlowast

lowastIndicates that genetic distance between the same genus does not exist it isshown for different genera

0700 0525 0350 0175 0000

CA

ZN

PZ

EF

RC

PHY

TA

Figure 5 UPGMA cluster analysis of fungal endophytes represent-ing seven medicinal plant species Plant codes are TA Tylophoraasthmatica RC Rubia cordifolia PZ Plumbago zeylanica PHYPhyllanthus amarus EF Eryngium foetidum CA Centella asiaticaand ZN Zingiber sp

PZ-EF formed the second cluster PHY RC and TA are foundoutside the cluster

4 Discussion

Medicinal plants are considered as a repository of ldquoendo-phytic microbesrdquo living in the internal tissues of plants Thequest for identifying novel bioactives from the endophyticfungi has resulted in the sampling of host plants such asherbs shrubs tree species and vines in unique places ofecological adaptations around the rainforests of the worldSuch niches harbor great species diversity unintervened by

human activities Efforts in this direction to sample plantslocated in the rainforests around the world with potentialethnomedicinal values have resulted in the isolation of fungalendophytes unique to a particular plant species with distinctbioactivity

41 Endophyte Colonization inMedicinal Species Themedic-inal plant species were sampled from the Talacauvery sub-cluster situated in the Kodagu District of Western Ghats ofSouthern India This region is among one of the 34 hot spotsof biodiversity Recently a proposal to include this biodiver-sity spot in the list of UNESCOHeritage cluster site is under-way (httpwwwatreeorgwg unesco whs) The natives aswell as the ethnic tribes inhabiting this region still dependon the plants as a source of medicine to cure some of theailments [12] Seven medicinal plant species assigned to sixplant families were selected for the study in natural popula-tions in two seasons from a single location from the studyarea stretching over an area of 25 kilometers Sampling wasconducted during monsoon and winter seasons as two ofthe herbaceous species E foetidum and Zingiber sp growonly till the second half of the year (June to December) andtheir nonavailability during summer (March toMay)makes itdifficult to consider the summer season for endophyticanalysis

From 5200 segments of plant materials a total of 1529isolates were obtained these were grouped into 31 taxaMycelia sterilia the fungal taxa that failed to sporulate werealso reported from this study This fungal group is prevalentin endophytic studies [24] The fungal endophytes wereanalyzed from four plant parts namely stem root rhizomeand inflorescence however their occurrence in root andinflorescence was investigated for few plant species only asthe phenology and sampling of plants never correlated withseasons The leaves were not considered for isolations sincesome of the plants were climbers and stragglers with delicatehairy surfaces and stringent surface sterilization techniqueswould render them unsuitable for plating on agar mediumRelative percentages of endophytic isolations from stemsegments were greater (8037) than isolations from roots(1922) Our results are supported by the earlier work ofHuang et al [25] on 29 traditional Chinese medicinal plantsthat fungal endophytes aremore frequent in stem tissues thanroots Among the fungal taxa coelomycete isolations weremore dominant than hyphomycetes and have been found inearlier studies in endophytes of tree species [26]

Endophytes such asColletotrichum PhomaAcremoniumChaetomium Botryodiplodia and Trichoderma were isolatedwith 119863 gt 20 Few fungal taxa that are less frequently iso-lated are Pestalotiopsis Penicillium islandicum Cladosporiumherbarum Alternaria alternata F graminearum Phomopsis

International Journal of Biodiversity 7

and Sphaeronema Colletotrichum spp are the most fre-quently encountered endophytic fungi [18] whereas Pestalo-tiopsis spp are well documented as endophytes of many rain-forest plants [27 28] tropical tree species namelyTerminaliaarjuna [29] Azadirachta indica [30] and many herbs andshrubs [25 31 32] It is necessary to screen newer plant speciesfor the isolation of fungal endophytes as Hawksworth andRossman [33] estimate that there are still millions of speciesof fungi yet to be identified Differences in the colonizationfrequencies of endophytes during two seasons were observedand more isolation during monsoon season is due to the factthat the slimy conidia of fungal spores are dispersed betterby rain splashes and germination of conidia is influenced byclimatic factors [34]

42 Host-Specificity of Fungal Endophytes We observed thatsome fungal taxa exhibited host-specificity a phenomenonoften associated with endophytes Three plant species Tasthmatica R cordifolia and P amarus were host-specificto endophytes The red pycnidial endophyte (TA-005) wasisolated from the stem fragments of T asthmatica onlysuggesting the host-specificity of this endophyte Pestalo-tiopsis guepinii was isolated from the stem segments of Tasthmatica It has been reported as an endophyte ofWollemianobilis growing in Sydney Australia [35] Heinesia rubiP islandicum and TA-005 are new reports of fungi asendophytes Host-specificity of endophytic fungi has beenobserved earlier for grasses [36] orchids [37] and forest treespecies [38 39] Recently Sun et al [40] reiterated the termldquohost-specificityrdquo as taxa that occur exclusively on a statedhost but not on other hosts in the same habitat [41] Ourstudies also indicate the host-specificity of endophytes as theplant species were sampled from a single habitat

43 Seasonal Diversity of Fungal Endophytes Diversityindices for fungal endophytes as analyzed by Shannon-Weiner (1198671) and Simpson (1119897) indices indicated differencesin seasonal variation and species richness High indices werenoted for T asthmatica (1198671 = 26) and P amarus (1198671 = 227)during monsoon and winter seasons respectivelyThe fungalspecies did not differ significantly between plant specieswhereas they differed between seasons (119875 lt 0001) Seasonalvariation in fungal isolates and colonization frequency hasbeen reported for many host plants [42 43] High coloniza-tion frequency as well as the species richness of endophyticfungi is limited to leaf segments rather than stem or bark seg-ments of host plants sampled from five medicinal species ofKudremukh Region of Western Ghats [44] Species richnessin our study is limited to stem fragments among the plantparts considered for analysis

Most studies on fungal endophytes in tropics haverevealed remarkable patterns of endophyte colonizationand estimates of diversity in foliages of forest tree speciesrepresenting various sites such as Panamanian Forest [45]and Iwokrama Forest Reserve Guyana [39] In the NilgiriBiosphere Reserve Western Ghats India 75 dicotyledonousspecies in three different tropical forest types were sampledto study foliar endophytes and diversity [10] The endophyte

diversity in forest types was limited due to loose hostaffiliations among endophytes Studies on foliar endophytesfrom the sampling of herbaceous and shrubby medicinalplant species have revealed differences in the colonizationrates as well as seasonal diversity inMalnad Region of BhadraWildlife Sanctuary in Southern India [32 46]

The present study provides firsthand information onthe diversity and seasonal influence on the colonizationfrequencies of endophytic fungi from selected medicinalplants from one of the subclusters of biodiversity hot spotsin the Western Ghats of Southern India Although theisolation and analysis of endophyte communities in herbsshrubs and trees are not uncommon each of the studies isunique with reference to number of hosts species of fungalendophytes and their specificityThe fungal endophytes havebeen subjected to fermentation studies and extracts are beingtested for biological activities

5 Conclusion

The study provides firsthand information on the diversityand seasonal influence on the colonization frequencies ofendophytic fungi from sevenmedicinal plants fromone of thesubclusters of biodiversity hot spots in the Western Ghats ofSouthern IndiaThe present investigation is the first isolationof endophytes from the medicinal species and their plantpartsThough isolation of endophytes has been accomplishedfrom various forest types and locations around the globeeach study is unique in documenting newer endophytic taxaWe are currently working on the fermentation of fungalendophytes to obtain newer antioxidants with therapeuticapplications

Conflict of Interests

The authors declare no conflict of interests regarding thepublication of this paper

Acknowledgments

This work was carried out with the financial assistancefrom the Department of Science amp Technology (DST)-SERCDivision Government of India under the Women Scien-tist Scheme [DST-WOS (A)] awarded to Monnanda Soma-iah Nalini (DST sanction no SRWOS (A)LS-762006 dt02082007) Monnanda Somaiah Nalini is thankful to theChairman DOS in Biotechnology for providing the neces-sary facilities for the completion of the project

References

[1] K Clay J Holah and J A Rudgers ldquoHerbivores cause a rapidincrease in hereditary symbiosis and alter plant communitycompositionrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 102 no 35 pp 12465ndash124702005

[2] P Alvarez-Loayza J F White Jr M S Torres et al ldquoLight con-verts endosymbiotic fungus to pathogen influencing seedling

8 International Journal of Biodiversity

survival and niche-space filling of a common tropical treeIriartea deltoideardquo PloS ONE vol 6 no 1 2011

[3] R Linnakoski H Puhakka-tarvainen andA Pappinen ldquoEndo-phytic fungi isolated from Khaya anthotheca in Ghanardquo FungalEcology vol 5 no 3 pp 298ndash308 2012

[4] J F White Jr and M S Torres ldquoIs plant endophyte-mediateddefensive mutualism the result of oxidative stress protectionrdquoPhysiologia Plantarum vol 138 no 4 pp 440ndash446 2010

[5] H-Y Li D-W Li C-M He Z-P Zhou T Mei and H-MXu ldquoDiversity and heavy metal tolerance of endophytic fungifrom six dominant plant species in a Pb-Zn mine wasteland inChinardquo Fungal Ecology vol 5 no 3 pp 309ndash315 2012

[6] A E Arnold ldquoUnderstanding the diversity of foliar endo-phytic fungi progress challenges and frontiersrdquo Fungal BiologyReviews vol 21 no 2-3 pp 51ndash66 2007

[7] K D Hyde and K Soytong ldquoThe fungal endophyte dilemmardquoFungal Diversity vol 33 pp 163ndash173 2008

[8] G Strobel and B Daisy ldquoBioprospecting for microbial endo-phytes and their natural productsrdquoMicrobiology and MolecularBiology Reviews vol 67 no 4 pp 491ndash502 2003

[9] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007

[10] T S Suryanarayanan T S Murali N Thirunavukkarasu M BGovinda Rajulu G Venkatesan and R Sukumar ldquoEndophyticfungal communities in woody perennials of three tropical foresttypes of the Western Ghats southern Indiardquo Biodiversity andConservation vol 20 no 5 pp 913ndash928 2011

[11] R A Mittermeier N Myers P R Gil and C G MittermeierHotspots Earthrsquos Biologically Richest and Most Endangered Ter-restrial Ecoregions Cemex Conservation International Wash-ington DC USA 2000

[12] R D Kshirsagar and N P Singh ldquoSome less known eth-nomedicinal uses from Mysore and Coorg districts Karnatakastate Indiardquo Journal of Ethnopharmacology vol 75 no 2-3 pp231ndash238 2001

[13] N YoganarasimhanMedicinal Plants of India Karnataka Inter-line Publishing Bangalore India 1996

[14] B Schulz U Wanke S Draeger and H J Aust ldquoEndophytesfrom herbaceous plants and shrubs effectiveness of surfacesterilization methodsrdquo Mycological Research vol 97 no 12 pp1447ndash1450 1993

[15] H Barnett and B Hunter Illustrated Genera of Imperfect FungiBurgess Publishing Minneapolis Minn USA 1998

[16] K H DomschW Gams and T AndersonCompendium of SoilFungi Academic Press New York NY USA 2003

[17] J F Leslie and B A Summerell The Fusarium LaboratoryManual Blackwell Publishing London UK 2006

[18] W Photita P W J Taylor R Ford K D Hyde and S Lumy-ong ldquoMorphological and molecular characterization of Col-letotrichum species from herbaceous plants inThailandrdquo FungalDiversity vol 18 pp 117ndash133 2005

[19] V Kumaresan and T S Suryanarayanan ldquoOccurrence and dis-tribution of endophytic fungi in a mangrove communityrdquoMycological Research vol 105 no 11 pp 1388ndash1391 2001

[20] V Kumaresan and T S Suryanarayanan ldquoEndophyte assem-blages in young mature and senescent leaves of Rhizophora

apiculata evidence for the role of endophytes inmangrove litterdegradationrdquo Fungal Diversity vol 9 pp 81ndash91 2002

[21] J A Ludwig and J F Reynolds Statistical Ecology A Primer onMethods and Computing John Wiley amp Sons New York NYUSA 1988

[22] M Nei ldquoGenetic distance between populationsrdquo AmericanNaturalist vol 106 no 949 pp 283ndash293 1972

[23] M P Miller Tools For Population Genetic Analyses (TFPGA)V L3 A Windows program For the Analysis of Allozyme AndmoleculAr Genetic data Department of Biological SciencesNorthern Arizona University Flagstaff Ariz USA 1997

[24] D C Lacap K D Hyde and E C Y Liew ldquoAn evaluationof the fungal rsquomorphotypersquo concept based on ribosomal DNAsequencesrdquo Fungal Diversity vol 12 pp 53ndash66 2003

[25] W Y Huang Y Z Cai K D Hyde H Corke and M SunldquoBiodiversity of endophytic fungi associated with 29 traditionalChinese medicinal plantsrdquo Fungal Diversity vol 33 pp 61ndash752008

[26] M V Tejesvi B Mahesh M S Nalini et al ldquoFungal endophyteassemblages from ethnopharmaceutically important medicinaltreesrdquo Canadian Journal of Microbiology vol 52 no 5 pp 427ndash435 2006

[27] N Raj Coelomycetous Anamorphs With Appendage BearingConidia Edwards Brothers Ann Harbor Michigan 1993

[28] G A Strobel ldquoMicrobial gifts from rain forestsrdquo CanadianJournal of Plant Pathology vol 24 no 1 pp 14ndash20 2002

[29] M V Tejesvi B Mahesh M S Nalini et al ldquoEndophytic fungalassemblages from inner bark and twig of Terminalia arjunaW amp A (Combretaceae)rdquo World Journal of Microbiology andBiotechnology vol 21 no 8-9 pp 1535ndash1540 2005

[30] B Mahesh M V Tejesvi M S Nalini et al ldquoEndophyticmycoflora of inner bark of Azadirachta indica A Jussrdquo CurrentScience vol 88 no 2 pp 218ndash219 2005

[31] K Rajagopal S Kalavathy S Kokila et al ldquoDiversity of fungalendophytes in few medicinal herbs of southern Indiardquo AsianJournal of Experimental Biological Sciences vol 1 pp 415ndash4182010

[32] Y L Krishnamurthy S B Naik and S Jayaram ldquoFungal com-munities in herbaceous medicinal plants from the malnadregion Southern IndiardquoMicrobes and Environments vol 23 no1 pp 24ndash28 2008

[33] D L Hawksworth and A Y Rossman ldquoWhere are all theundescribed fungirdquo Phytopathology vol 87 no 9 pp 888ndash8911997

[34] D Wilson and G C CarrolL ldquoInfection studies of Disculaquercina an endophyte of Quercus garryanardquo Mycologia vol86 no 5 pp 635ndash647 1994

[35] G A Strobel W M Hess J-Y Li et al ldquoPestalotiopsis guepiniia taxol-producing endophyte of the wollemi pine Wollemianobilisrdquo Australian Journal of Botany vol 45 no 6 pp 1073ndash1082 1997

[36] K Clay ldquoFungal endophytes of grassesrdquo Annual Review ofEcology and Systematics vol 21 no 1 pp 275ndash297 1990

[37] P Bayman L L Lebron R L Tremblay and D J Lodge ldquoVari-ation in endophytic fungi from roots and leaves of Lepanthes(Orchidaceae)rdquoNewPhytologist vol 135 no 1 pp 143ndash149 1997

[38] A EArnold ZMaynard andG SGilbert ldquoFungal endophytesin dicotyledonous neotropical trees patterns of abundance and

International Journal of Biodiversity 9

diversityrdquo Mycological Research vol 105 no 12 pp 1502ndash15072001

[39] P F Cannon andCM Simmons ldquoDiversity andhost preferenceof leaf endophytic fungi in the Iwokrama Forest ReserveGuyanardquoMycologia vol 94 no 2 pp 210ndash220 2002

[40] X Sun Q Ding K D Hyde and L D Guo ldquoCommunitystructure and preference of endophytic fungi of three woodyplants in a mixed forestrdquo Fungal Ecology vol 5 no 5 pp 624ndash632 2012

[41] P HollidayADictionary of Plant Pathology CambridgeUniver-sity Press Cambridge UK 1998

[42] J Collado G Platas and F Pelaez ldquoIdentification of an endo-phytic Nodulisporium sp from Quercus ilex in central Spainas the anamorph of Biscogniauxia mediterranea by rDNAsequence analysis and effect of different ecological factors ondistribution of the fungusrdquo Mycologia vol 93 no 5 pp 875ndash886 2001

[43] X-X Gao H Zhou D-Y Xu C-H Yu Y-Q Chen and L-HQu ldquoHigh diversity of endophytic fungi from the pharmaceuti-cal plant Heterosmilax japonica Kunth revealed by cultivation-independent approachrdquo FEMS Microbiology Letters vol 249no 2 pp 255ndash266 2005

[44] N S Raviraja ldquoFungal endophytes in five medicinal plantspecies from Kudremukh Range Western Ghats of IndiardquoJournal of Basic Microbiology vol 45 no 3 pp 230ndash235 2005

[45] A E Arnold Z Maynard G S Gilbert P D Coley and T AKursar ldquoAre tropical fungal endophytes hyperdiverserdquo EcologyLetters vol 3 no 4 pp 267ndash274 2000

[46] B S Naik J Shashikala and Y L Krishnamurthy ldquoDiversityof fungal endophytes in shrubby medicinal plants of Malnadregion Western Ghats Southern Indiardquo Fungal Ecology vol 1no 2-3 pp 89ndash93 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

6 International Journal of Biodiversity

Table 4 ANOVA table of seasonal variation of endophytic fungi analyzed from seven medicinal plant species

Sum of squares Degrees of freedom Mean2119865 Significance

Between seasons 119319 26 458919 34382 0001Within seasons 1001062 75 13346 mdash mdashTotal 1293296 101 mdash mdash mdash

Table 5 Neirsquos genetic distance of plant species analyzed for endo-phytic fungi

TA RC PZ PHY EF CA ZNTA lowastlowastlowast 07885 05008 06061 04055 07885 09116RC lowastlowastlowast 05008 07239 04055 03610 06061PZ lowastlowastlowast 06633 03610 05008 04520PHY lowastlowastlowast 04520 05008 06633EF lowastlowastlowast 04055 03610CA lowastlowastlowast 03185ZN lowastlowastlowast

lowastIndicates that genetic distance between the same genus does not exist it isshown for different genera

0700 0525 0350 0175 0000

CA

ZN

PZ

EF

RC

PHY

TA

Figure 5 UPGMA cluster analysis of fungal endophytes represent-ing seven medicinal plant species Plant codes are TA Tylophoraasthmatica RC Rubia cordifolia PZ Plumbago zeylanica PHYPhyllanthus amarus EF Eryngium foetidum CA Centella asiaticaand ZN Zingiber sp

PZ-EF formed the second cluster PHY RC and TA are foundoutside the cluster

4 Discussion

Medicinal plants are considered as a repository of ldquoendo-phytic microbesrdquo living in the internal tissues of plants Thequest for identifying novel bioactives from the endophyticfungi has resulted in the sampling of host plants such asherbs shrubs tree species and vines in unique places ofecological adaptations around the rainforests of the worldSuch niches harbor great species diversity unintervened by

human activities Efforts in this direction to sample plantslocated in the rainforests around the world with potentialethnomedicinal values have resulted in the isolation of fungalendophytes unique to a particular plant species with distinctbioactivity

41 Endophyte Colonization inMedicinal Species Themedic-inal plant species were sampled from the Talacauvery sub-cluster situated in the Kodagu District of Western Ghats ofSouthern India This region is among one of the 34 hot spotsof biodiversity Recently a proposal to include this biodiver-sity spot in the list of UNESCOHeritage cluster site is under-way (httpwwwatreeorgwg unesco whs) The natives aswell as the ethnic tribes inhabiting this region still dependon the plants as a source of medicine to cure some of theailments [12] Seven medicinal plant species assigned to sixplant families were selected for the study in natural popula-tions in two seasons from a single location from the studyarea stretching over an area of 25 kilometers Sampling wasconducted during monsoon and winter seasons as two ofthe herbaceous species E foetidum and Zingiber sp growonly till the second half of the year (June to December) andtheir nonavailability during summer (March toMay)makes itdifficult to consider the summer season for endophyticanalysis

From 5200 segments of plant materials a total of 1529isolates were obtained these were grouped into 31 taxaMycelia sterilia the fungal taxa that failed to sporulate werealso reported from this study This fungal group is prevalentin endophytic studies [24] The fungal endophytes wereanalyzed from four plant parts namely stem root rhizomeand inflorescence however their occurrence in root andinflorescence was investigated for few plant species only asthe phenology and sampling of plants never correlated withseasons The leaves were not considered for isolations sincesome of the plants were climbers and stragglers with delicatehairy surfaces and stringent surface sterilization techniqueswould render them unsuitable for plating on agar mediumRelative percentages of endophytic isolations from stemsegments were greater (8037) than isolations from roots(1922) Our results are supported by the earlier work ofHuang et al [25] on 29 traditional Chinese medicinal plantsthat fungal endophytes aremore frequent in stem tissues thanroots Among the fungal taxa coelomycete isolations weremore dominant than hyphomycetes and have been found inearlier studies in endophytes of tree species [26]

Endophytes such asColletotrichum PhomaAcremoniumChaetomium Botryodiplodia and Trichoderma were isolatedwith 119863 gt 20 Few fungal taxa that are less frequently iso-lated are Pestalotiopsis Penicillium islandicum Cladosporiumherbarum Alternaria alternata F graminearum Phomopsis

International Journal of Biodiversity 7

and Sphaeronema Colletotrichum spp are the most fre-quently encountered endophytic fungi [18] whereas Pestalo-tiopsis spp are well documented as endophytes of many rain-forest plants [27 28] tropical tree species namelyTerminaliaarjuna [29] Azadirachta indica [30] and many herbs andshrubs [25 31 32] It is necessary to screen newer plant speciesfor the isolation of fungal endophytes as Hawksworth andRossman [33] estimate that there are still millions of speciesof fungi yet to be identified Differences in the colonizationfrequencies of endophytes during two seasons were observedand more isolation during monsoon season is due to the factthat the slimy conidia of fungal spores are dispersed betterby rain splashes and germination of conidia is influenced byclimatic factors [34]

42 Host-Specificity of Fungal Endophytes We observed thatsome fungal taxa exhibited host-specificity a phenomenonoften associated with endophytes Three plant species Tasthmatica R cordifolia and P amarus were host-specificto endophytes The red pycnidial endophyte (TA-005) wasisolated from the stem fragments of T asthmatica onlysuggesting the host-specificity of this endophyte Pestalo-tiopsis guepinii was isolated from the stem segments of Tasthmatica It has been reported as an endophyte ofWollemianobilis growing in Sydney Australia [35] Heinesia rubiP islandicum and TA-005 are new reports of fungi asendophytes Host-specificity of endophytic fungi has beenobserved earlier for grasses [36] orchids [37] and forest treespecies [38 39] Recently Sun et al [40] reiterated the termldquohost-specificityrdquo as taxa that occur exclusively on a statedhost but not on other hosts in the same habitat [41] Ourstudies also indicate the host-specificity of endophytes as theplant species were sampled from a single habitat

43 Seasonal Diversity of Fungal Endophytes Diversityindices for fungal endophytes as analyzed by Shannon-Weiner (1198671) and Simpson (1119897) indices indicated differencesin seasonal variation and species richness High indices werenoted for T asthmatica (1198671 = 26) and P amarus (1198671 = 227)during monsoon and winter seasons respectivelyThe fungalspecies did not differ significantly between plant specieswhereas they differed between seasons (119875 lt 0001) Seasonalvariation in fungal isolates and colonization frequency hasbeen reported for many host plants [42 43] High coloniza-tion frequency as well as the species richness of endophyticfungi is limited to leaf segments rather than stem or bark seg-ments of host plants sampled from five medicinal species ofKudremukh Region of Western Ghats [44] Species richnessin our study is limited to stem fragments among the plantparts considered for analysis

Most studies on fungal endophytes in tropics haverevealed remarkable patterns of endophyte colonizationand estimates of diversity in foliages of forest tree speciesrepresenting various sites such as Panamanian Forest [45]and Iwokrama Forest Reserve Guyana [39] In the NilgiriBiosphere Reserve Western Ghats India 75 dicotyledonousspecies in three different tropical forest types were sampledto study foliar endophytes and diversity [10] The endophyte

diversity in forest types was limited due to loose hostaffiliations among endophytes Studies on foliar endophytesfrom the sampling of herbaceous and shrubby medicinalplant species have revealed differences in the colonizationrates as well as seasonal diversity inMalnad Region of BhadraWildlife Sanctuary in Southern India [32 46]

The present study provides firsthand information onthe diversity and seasonal influence on the colonizationfrequencies of endophytic fungi from selected medicinalplants from one of the subclusters of biodiversity hot spotsin the Western Ghats of Southern India Although theisolation and analysis of endophyte communities in herbsshrubs and trees are not uncommon each of the studies isunique with reference to number of hosts species of fungalendophytes and their specificityThe fungal endophytes havebeen subjected to fermentation studies and extracts are beingtested for biological activities

5 Conclusion

The study provides firsthand information on the diversityand seasonal influence on the colonization frequencies ofendophytic fungi from sevenmedicinal plants fromone of thesubclusters of biodiversity hot spots in the Western Ghats ofSouthern IndiaThe present investigation is the first isolationof endophytes from the medicinal species and their plantpartsThough isolation of endophytes has been accomplishedfrom various forest types and locations around the globeeach study is unique in documenting newer endophytic taxaWe are currently working on the fermentation of fungalendophytes to obtain newer antioxidants with therapeuticapplications

Conflict of Interests

The authors declare no conflict of interests regarding thepublication of this paper

Acknowledgments

This work was carried out with the financial assistancefrom the Department of Science amp Technology (DST)-SERCDivision Government of India under the Women Scien-tist Scheme [DST-WOS (A)] awarded to Monnanda Soma-iah Nalini (DST sanction no SRWOS (A)LS-762006 dt02082007) Monnanda Somaiah Nalini is thankful to theChairman DOS in Biotechnology for providing the neces-sary facilities for the completion of the project

References

[1] K Clay J Holah and J A Rudgers ldquoHerbivores cause a rapidincrease in hereditary symbiosis and alter plant communitycompositionrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 102 no 35 pp 12465ndash124702005

[2] P Alvarez-Loayza J F White Jr M S Torres et al ldquoLight con-verts endosymbiotic fungus to pathogen influencing seedling

8 International Journal of Biodiversity

survival and niche-space filling of a common tropical treeIriartea deltoideardquo PloS ONE vol 6 no 1 2011

[3] R Linnakoski H Puhakka-tarvainen andA Pappinen ldquoEndo-phytic fungi isolated from Khaya anthotheca in Ghanardquo FungalEcology vol 5 no 3 pp 298ndash308 2012

[4] J F White Jr and M S Torres ldquoIs plant endophyte-mediateddefensive mutualism the result of oxidative stress protectionrdquoPhysiologia Plantarum vol 138 no 4 pp 440ndash446 2010

[5] H-Y Li D-W Li C-M He Z-P Zhou T Mei and H-MXu ldquoDiversity and heavy metal tolerance of endophytic fungifrom six dominant plant species in a Pb-Zn mine wasteland inChinardquo Fungal Ecology vol 5 no 3 pp 309ndash315 2012

[6] A E Arnold ldquoUnderstanding the diversity of foliar endo-phytic fungi progress challenges and frontiersrdquo Fungal BiologyReviews vol 21 no 2-3 pp 51ndash66 2007

[7] K D Hyde and K Soytong ldquoThe fungal endophyte dilemmardquoFungal Diversity vol 33 pp 163ndash173 2008

[8] G Strobel and B Daisy ldquoBioprospecting for microbial endo-phytes and their natural productsrdquoMicrobiology and MolecularBiology Reviews vol 67 no 4 pp 491ndash502 2003

[9] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007

[10] T S Suryanarayanan T S Murali N Thirunavukkarasu M BGovinda Rajulu G Venkatesan and R Sukumar ldquoEndophyticfungal communities in woody perennials of three tropical foresttypes of the Western Ghats southern Indiardquo Biodiversity andConservation vol 20 no 5 pp 913ndash928 2011

[11] R A Mittermeier N Myers P R Gil and C G MittermeierHotspots Earthrsquos Biologically Richest and Most Endangered Ter-restrial Ecoregions Cemex Conservation International Wash-ington DC USA 2000

[12] R D Kshirsagar and N P Singh ldquoSome less known eth-nomedicinal uses from Mysore and Coorg districts Karnatakastate Indiardquo Journal of Ethnopharmacology vol 75 no 2-3 pp231ndash238 2001

[13] N YoganarasimhanMedicinal Plants of India Karnataka Inter-line Publishing Bangalore India 1996

[14] B Schulz U Wanke S Draeger and H J Aust ldquoEndophytesfrom herbaceous plants and shrubs effectiveness of surfacesterilization methodsrdquo Mycological Research vol 97 no 12 pp1447ndash1450 1993

[15] H Barnett and B Hunter Illustrated Genera of Imperfect FungiBurgess Publishing Minneapolis Minn USA 1998

[16] K H DomschW Gams and T AndersonCompendium of SoilFungi Academic Press New York NY USA 2003

[17] J F Leslie and B A Summerell The Fusarium LaboratoryManual Blackwell Publishing London UK 2006

[18] W Photita P W J Taylor R Ford K D Hyde and S Lumy-ong ldquoMorphological and molecular characterization of Col-letotrichum species from herbaceous plants inThailandrdquo FungalDiversity vol 18 pp 117ndash133 2005

[19] V Kumaresan and T S Suryanarayanan ldquoOccurrence and dis-tribution of endophytic fungi in a mangrove communityrdquoMycological Research vol 105 no 11 pp 1388ndash1391 2001

[20] V Kumaresan and T S Suryanarayanan ldquoEndophyte assem-blages in young mature and senescent leaves of Rhizophora

apiculata evidence for the role of endophytes inmangrove litterdegradationrdquo Fungal Diversity vol 9 pp 81ndash91 2002

[21] J A Ludwig and J F Reynolds Statistical Ecology A Primer onMethods and Computing John Wiley amp Sons New York NYUSA 1988

[22] M Nei ldquoGenetic distance between populationsrdquo AmericanNaturalist vol 106 no 949 pp 283ndash293 1972

[23] M P Miller Tools For Population Genetic Analyses (TFPGA)V L3 A Windows program For the Analysis of Allozyme AndmoleculAr Genetic data Department of Biological SciencesNorthern Arizona University Flagstaff Ariz USA 1997

[24] D C Lacap K D Hyde and E C Y Liew ldquoAn evaluationof the fungal rsquomorphotypersquo concept based on ribosomal DNAsequencesrdquo Fungal Diversity vol 12 pp 53ndash66 2003

[25] W Y Huang Y Z Cai K D Hyde H Corke and M SunldquoBiodiversity of endophytic fungi associated with 29 traditionalChinese medicinal plantsrdquo Fungal Diversity vol 33 pp 61ndash752008

[26] M V Tejesvi B Mahesh M S Nalini et al ldquoFungal endophyteassemblages from ethnopharmaceutically important medicinaltreesrdquo Canadian Journal of Microbiology vol 52 no 5 pp 427ndash435 2006

[27] N Raj Coelomycetous Anamorphs With Appendage BearingConidia Edwards Brothers Ann Harbor Michigan 1993

[28] G A Strobel ldquoMicrobial gifts from rain forestsrdquo CanadianJournal of Plant Pathology vol 24 no 1 pp 14ndash20 2002

[29] M V Tejesvi B Mahesh M S Nalini et al ldquoEndophytic fungalassemblages from inner bark and twig of Terminalia arjunaW amp A (Combretaceae)rdquo World Journal of Microbiology andBiotechnology vol 21 no 8-9 pp 1535ndash1540 2005

[30] B Mahesh M V Tejesvi M S Nalini et al ldquoEndophyticmycoflora of inner bark of Azadirachta indica A Jussrdquo CurrentScience vol 88 no 2 pp 218ndash219 2005

[31] K Rajagopal S Kalavathy S Kokila et al ldquoDiversity of fungalendophytes in few medicinal herbs of southern Indiardquo AsianJournal of Experimental Biological Sciences vol 1 pp 415ndash4182010

[32] Y L Krishnamurthy S B Naik and S Jayaram ldquoFungal com-munities in herbaceous medicinal plants from the malnadregion Southern IndiardquoMicrobes and Environments vol 23 no1 pp 24ndash28 2008

[33] D L Hawksworth and A Y Rossman ldquoWhere are all theundescribed fungirdquo Phytopathology vol 87 no 9 pp 888ndash8911997

[34] D Wilson and G C CarrolL ldquoInfection studies of Disculaquercina an endophyte of Quercus garryanardquo Mycologia vol86 no 5 pp 635ndash647 1994

[35] G A Strobel W M Hess J-Y Li et al ldquoPestalotiopsis guepiniia taxol-producing endophyte of the wollemi pine Wollemianobilisrdquo Australian Journal of Botany vol 45 no 6 pp 1073ndash1082 1997

[36] K Clay ldquoFungal endophytes of grassesrdquo Annual Review ofEcology and Systematics vol 21 no 1 pp 275ndash297 1990

[37] P Bayman L L Lebron R L Tremblay and D J Lodge ldquoVari-ation in endophytic fungi from roots and leaves of Lepanthes(Orchidaceae)rdquoNewPhytologist vol 135 no 1 pp 143ndash149 1997

[38] A EArnold ZMaynard andG SGilbert ldquoFungal endophytesin dicotyledonous neotropical trees patterns of abundance and

International Journal of Biodiversity 9

diversityrdquo Mycological Research vol 105 no 12 pp 1502ndash15072001

[39] P F Cannon andCM Simmons ldquoDiversity andhost preferenceof leaf endophytic fungi in the Iwokrama Forest ReserveGuyanardquoMycologia vol 94 no 2 pp 210ndash220 2002

[40] X Sun Q Ding K D Hyde and L D Guo ldquoCommunitystructure and preference of endophytic fungi of three woodyplants in a mixed forestrdquo Fungal Ecology vol 5 no 5 pp 624ndash632 2012

[41] P HollidayADictionary of Plant Pathology CambridgeUniver-sity Press Cambridge UK 1998

[42] J Collado G Platas and F Pelaez ldquoIdentification of an endo-phytic Nodulisporium sp from Quercus ilex in central Spainas the anamorph of Biscogniauxia mediterranea by rDNAsequence analysis and effect of different ecological factors ondistribution of the fungusrdquo Mycologia vol 93 no 5 pp 875ndash886 2001

[43] X-X Gao H Zhou D-Y Xu C-H Yu Y-Q Chen and L-HQu ldquoHigh diversity of endophytic fungi from the pharmaceuti-cal plant Heterosmilax japonica Kunth revealed by cultivation-independent approachrdquo FEMS Microbiology Letters vol 249no 2 pp 255ndash266 2005

[44] N S Raviraja ldquoFungal endophytes in five medicinal plantspecies from Kudremukh Range Western Ghats of IndiardquoJournal of Basic Microbiology vol 45 no 3 pp 230ndash235 2005

[45] A E Arnold Z Maynard G S Gilbert P D Coley and T AKursar ldquoAre tropical fungal endophytes hyperdiverserdquo EcologyLetters vol 3 no 4 pp 267ndash274 2000

[46] B S Naik J Shashikala and Y L Krishnamurthy ldquoDiversityof fungal endophytes in shrubby medicinal plants of Malnadregion Western Ghats Southern Indiardquo Fungal Ecology vol 1no 2-3 pp 89ndash93 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

International Journal of Biodiversity 7

and Sphaeronema Colletotrichum spp are the most fre-quently encountered endophytic fungi [18] whereas Pestalo-tiopsis spp are well documented as endophytes of many rain-forest plants [27 28] tropical tree species namelyTerminaliaarjuna [29] Azadirachta indica [30] and many herbs andshrubs [25 31 32] It is necessary to screen newer plant speciesfor the isolation of fungal endophytes as Hawksworth andRossman [33] estimate that there are still millions of speciesof fungi yet to be identified Differences in the colonizationfrequencies of endophytes during two seasons were observedand more isolation during monsoon season is due to the factthat the slimy conidia of fungal spores are dispersed betterby rain splashes and germination of conidia is influenced byclimatic factors [34]

42 Host-Specificity of Fungal Endophytes We observed thatsome fungal taxa exhibited host-specificity a phenomenonoften associated with endophytes Three plant species Tasthmatica R cordifolia and P amarus were host-specificto endophytes The red pycnidial endophyte (TA-005) wasisolated from the stem fragments of T asthmatica onlysuggesting the host-specificity of this endophyte Pestalo-tiopsis guepinii was isolated from the stem segments of Tasthmatica It has been reported as an endophyte ofWollemianobilis growing in Sydney Australia [35] Heinesia rubiP islandicum and TA-005 are new reports of fungi asendophytes Host-specificity of endophytic fungi has beenobserved earlier for grasses [36] orchids [37] and forest treespecies [38 39] Recently Sun et al [40] reiterated the termldquohost-specificityrdquo as taxa that occur exclusively on a statedhost but not on other hosts in the same habitat [41] Ourstudies also indicate the host-specificity of endophytes as theplant species were sampled from a single habitat

43 Seasonal Diversity of Fungal Endophytes Diversityindices for fungal endophytes as analyzed by Shannon-Weiner (1198671) and Simpson (1119897) indices indicated differencesin seasonal variation and species richness High indices werenoted for T asthmatica (1198671 = 26) and P amarus (1198671 = 227)during monsoon and winter seasons respectivelyThe fungalspecies did not differ significantly between plant specieswhereas they differed between seasons (119875 lt 0001) Seasonalvariation in fungal isolates and colonization frequency hasbeen reported for many host plants [42 43] High coloniza-tion frequency as well as the species richness of endophyticfungi is limited to leaf segments rather than stem or bark seg-ments of host plants sampled from five medicinal species ofKudremukh Region of Western Ghats [44] Species richnessin our study is limited to stem fragments among the plantparts considered for analysis

Most studies on fungal endophytes in tropics haverevealed remarkable patterns of endophyte colonizationand estimates of diversity in foliages of forest tree speciesrepresenting various sites such as Panamanian Forest [45]and Iwokrama Forest Reserve Guyana [39] In the NilgiriBiosphere Reserve Western Ghats India 75 dicotyledonousspecies in three different tropical forest types were sampledto study foliar endophytes and diversity [10] The endophyte

diversity in forest types was limited due to loose hostaffiliations among endophytes Studies on foliar endophytesfrom the sampling of herbaceous and shrubby medicinalplant species have revealed differences in the colonizationrates as well as seasonal diversity inMalnad Region of BhadraWildlife Sanctuary in Southern India [32 46]

The present study provides firsthand information onthe diversity and seasonal influence on the colonizationfrequencies of endophytic fungi from selected medicinalplants from one of the subclusters of biodiversity hot spotsin the Western Ghats of Southern India Although theisolation and analysis of endophyte communities in herbsshrubs and trees are not uncommon each of the studies isunique with reference to number of hosts species of fungalendophytes and their specificityThe fungal endophytes havebeen subjected to fermentation studies and extracts are beingtested for biological activities

5 Conclusion

The study provides firsthand information on the diversityand seasonal influence on the colonization frequencies ofendophytic fungi from sevenmedicinal plants fromone of thesubclusters of biodiversity hot spots in the Western Ghats ofSouthern IndiaThe present investigation is the first isolationof endophytes from the medicinal species and their plantpartsThough isolation of endophytes has been accomplishedfrom various forest types and locations around the globeeach study is unique in documenting newer endophytic taxaWe are currently working on the fermentation of fungalendophytes to obtain newer antioxidants with therapeuticapplications

Conflict of Interests

The authors declare no conflict of interests regarding thepublication of this paper

Acknowledgments

This work was carried out with the financial assistancefrom the Department of Science amp Technology (DST)-SERCDivision Government of India under the Women Scien-tist Scheme [DST-WOS (A)] awarded to Monnanda Soma-iah Nalini (DST sanction no SRWOS (A)LS-762006 dt02082007) Monnanda Somaiah Nalini is thankful to theChairman DOS in Biotechnology for providing the neces-sary facilities for the completion of the project

References

[1] K Clay J Holah and J A Rudgers ldquoHerbivores cause a rapidincrease in hereditary symbiosis and alter plant communitycompositionrdquo Proceedings of the National Academy of Sciencesof the United States of America vol 102 no 35 pp 12465ndash124702005

[2] P Alvarez-Loayza J F White Jr M S Torres et al ldquoLight con-verts endosymbiotic fungus to pathogen influencing seedling

8 International Journal of Biodiversity

survival and niche-space filling of a common tropical treeIriartea deltoideardquo PloS ONE vol 6 no 1 2011

[3] R Linnakoski H Puhakka-tarvainen andA Pappinen ldquoEndo-phytic fungi isolated from Khaya anthotheca in Ghanardquo FungalEcology vol 5 no 3 pp 298ndash308 2012

[4] J F White Jr and M S Torres ldquoIs plant endophyte-mediateddefensive mutualism the result of oxidative stress protectionrdquoPhysiologia Plantarum vol 138 no 4 pp 440ndash446 2010

[5] H-Y Li D-W Li C-M He Z-P Zhou T Mei and H-MXu ldquoDiversity and heavy metal tolerance of endophytic fungifrom six dominant plant species in a Pb-Zn mine wasteland inChinardquo Fungal Ecology vol 5 no 3 pp 309ndash315 2012

[6] A E Arnold ldquoUnderstanding the diversity of foliar endo-phytic fungi progress challenges and frontiersrdquo Fungal BiologyReviews vol 21 no 2-3 pp 51ndash66 2007

[7] K D Hyde and K Soytong ldquoThe fungal endophyte dilemmardquoFungal Diversity vol 33 pp 163ndash173 2008

[8] G Strobel and B Daisy ldquoBioprospecting for microbial endo-phytes and their natural productsrdquoMicrobiology and MolecularBiology Reviews vol 67 no 4 pp 491ndash502 2003

[9] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007

[10] T S Suryanarayanan T S Murali N Thirunavukkarasu M BGovinda Rajulu G Venkatesan and R Sukumar ldquoEndophyticfungal communities in woody perennials of three tropical foresttypes of the Western Ghats southern Indiardquo Biodiversity andConservation vol 20 no 5 pp 913ndash928 2011

[11] R A Mittermeier N Myers P R Gil and C G MittermeierHotspots Earthrsquos Biologically Richest and Most Endangered Ter-restrial Ecoregions Cemex Conservation International Wash-ington DC USA 2000

[12] R D Kshirsagar and N P Singh ldquoSome less known eth-nomedicinal uses from Mysore and Coorg districts Karnatakastate Indiardquo Journal of Ethnopharmacology vol 75 no 2-3 pp231ndash238 2001

[13] N YoganarasimhanMedicinal Plants of India Karnataka Inter-line Publishing Bangalore India 1996

[14] B Schulz U Wanke S Draeger and H J Aust ldquoEndophytesfrom herbaceous plants and shrubs effectiveness of surfacesterilization methodsrdquo Mycological Research vol 97 no 12 pp1447ndash1450 1993

[15] H Barnett and B Hunter Illustrated Genera of Imperfect FungiBurgess Publishing Minneapolis Minn USA 1998

[16] K H DomschW Gams and T AndersonCompendium of SoilFungi Academic Press New York NY USA 2003

[17] J F Leslie and B A Summerell The Fusarium LaboratoryManual Blackwell Publishing London UK 2006

[18] W Photita P W J Taylor R Ford K D Hyde and S Lumy-ong ldquoMorphological and molecular characterization of Col-letotrichum species from herbaceous plants inThailandrdquo FungalDiversity vol 18 pp 117ndash133 2005

[19] V Kumaresan and T S Suryanarayanan ldquoOccurrence and dis-tribution of endophytic fungi in a mangrove communityrdquoMycological Research vol 105 no 11 pp 1388ndash1391 2001

[20] V Kumaresan and T S Suryanarayanan ldquoEndophyte assem-blages in young mature and senescent leaves of Rhizophora

apiculata evidence for the role of endophytes inmangrove litterdegradationrdquo Fungal Diversity vol 9 pp 81ndash91 2002

[21] J A Ludwig and J F Reynolds Statistical Ecology A Primer onMethods and Computing John Wiley amp Sons New York NYUSA 1988

[22] M Nei ldquoGenetic distance between populationsrdquo AmericanNaturalist vol 106 no 949 pp 283ndash293 1972

[23] M P Miller Tools For Population Genetic Analyses (TFPGA)V L3 A Windows program For the Analysis of Allozyme AndmoleculAr Genetic data Department of Biological SciencesNorthern Arizona University Flagstaff Ariz USA 1997

[24] D C Lacap K D Hyde and E C Y Liew ldquoAn evaluationof the fungal rsquomorphotypersquo concept based on ribosomal DNAsequencesrdquo Fungal Diversity vol 12 pp 53ndash66 2003

[25] W Y Huang Y Z Cai K D Hyde H Corke and M SunldquoBiodiversity of endophytic fungi associated with 29 traditionalChinese medicinal plantsrdquo Fungal Diversity vol 33 pp 61ndash752008

[26] M V Tejesvi B Mahesh M S Nalini et al ldquoFungal endophyteassemblages from ethnopharmaceutically important medicinaltreesrdquo Canadian Journal of Microbiology vol 52 no 5 pp 427ndash435 2006

[27] N Raj Coelomycetous Anamorphs With Appendage BearingConidia Edwards Brothers Ann Harbor Michigan 1993

[28] G A Strobel ldquoMicrobial gifts from rain forestsrdquo CanadianJournal of Plant Pathology vol 24 no 1 pp 14ndash20 2002

[29] M V Tejesvi B Mahesh M S Nalini et al ldquoEndophytic fungalassemblages from inner bark and twig of Terminalia arjunaW amp A (Combretaceae)rdquo World Journal of Microbiology andBiotechnology vol 21 no 8-9 pp 1535ndash1540 2005

[30] B Mahesh M V Tejesvi M S Nalini et al ldquoEndophyticmycoflora of inner bark of Azadirachta indica A Jussrdquo CurrentScience vol 88 no 2 pp 218ndash219 2005

[31] K Rajagopal S Kalavathy S Kokila et al ldquoDiversity of fungalendophytes in few medicinal herbs of southern Indiardquo AsianJournal of Experimental Biological Sciences vol 1 pp 415ndash4182010

[32] Y L Krishnamurthy S B Naik and S Jayaram ldquoFungal com-munities in herbaceous medicinal plants from the malnadregion Southern IndiardquoMicrobes and Environments vol 23 no1 pp 24ndash28 2008

[33] D L Hawksworth and A Y Rossman ldquoWhere are all theundescribed fungirdquo Phytopathology vol 87 no 9 pp 888ndash8911997

[34] D Wilson and G C CarrolL ldquoInfection studies of Disculaquercina an endophyte of Quercus garryanardquo Mycologia vol86 no 5 pp 635ndash647 1994

[35] G A Strobel W M Hess J-Y Li et al ldquoPestalotiopsis guepiniia taxol-producing endophyte of the wollemi pine Wollemianobilisrdquo Australian Journal of Botany vol 45 no 6 pp 1073ndash1082 1997

[36] K Clay ldquoFungal endophytes of grassesrdquo Annual Review ofEcology and Systematics vol 21 no 1 pp 275ndash297 1990

[37] P Bayman L L Lebron R L Tremblay and D J Lodge ldquoVari-ation in endophytic fungi from roots and leaves of Lepanthes(Orchidaceae)rdquoNewPhytologist vol 135 no 1 pp 143ndash149 1997

[38] A EArnold ZMaynard andG SGilbert ldquoFungal endophytesin dicotyledonous neotropical trees patterns of abundance and

International Journal of Biodiversity 9

diversityrdquo Mycological Research vol 105 no 12 pp 1502ndash15072001

[39] P F Cannon andCM Simmons ldquoDiversity andhost preferenceof leaf endophytic fungi in the Iwokrama Forest ReserveGuyanardquoMycologia vol 94 no 2 pp 210ndash220 2002

[40] X Sun Q Ding K D Hyde and L D Guo ldquoCommunitystructure and preference of endophytic fungi of three woodyplants in a mixed forestrdquo Fungal Ecology vol 5 no 5 pp 624ndash632 2012

[41] P HollidayADictionary of Plant Pathology CambridgeUniver-sity Press Cambridge UK 1998

[42] J Collado G Platas and F Pelaez ldquoIdentification of an endo-phytic Nodulisporium sp from Quercus ilex in central Spainas the anamorph of Biscogniauxia mediterranea by rDNAsequence analysis and effect of different ecological factors ondistribution of the fungusrdquo Mycologia vol 93 no 5 pp 875ndash886 2001

[43] X-X Gao H Zhou D-Y Xu C-H Yu Y-Q Chen and L-HQu ldquoHigh diversity of endophytic fungi from the pharmaceuti-cal plant Heterosmilax japonica Kunth revealed by cultivation-independent approachrdquo FEMS Microbiology Letters vol 249no 2 pp 255ndash266 2005

[44] N S Raviraja ldquoFungal endophytes in five medicinal plantspecies from Kudremukh Range Western Ghats of IndiardquoJournal of Basic Microbiology vol 45 no 3 pp 230ndash235 2005

[45] A E Arnold Z Maynard G S Gilbert P D Coley and T AKursar ldquoAre tropical fungal endophytes hyperdiverserdquo EcologyLetters vol 3 no 4 pp 267ndash274 2000

[46] B S Naik J Shashikala and Y L Krishnamurthy ldquoDiversityof fungal endophytes in shrubby medicinal plants of Malnadregion Western Ghats Southern Indiardquo Fungal Ecology vol 1no 2-3 pp 89ndash93 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

8 International Journal of Biodiversity

survival and niche-space filling of a common tropical treeIriartea deltoideardquo PloS ONE vol 6 no 1 2011

[3] R Linnakoski H Puhakka-tarvainen andA Pappinen ldquoEndo-phytic fungi isolated from Khaya anthotheca in Ghanardquo FungalEcology vol 5 no 3 pp 298ndash308 2012

[4] J F White Jr and M S Torres ldquoIs plant endophyte-mediateddefensive mutualism the result of oxidative stress protectionrdquoPhysiologia Plantarum vol 138 no 4 pp 440ndash446 2010

[5] H-Y Li D-W Li C-M He Z-P Zhou T Mei and H-MXu ldquoDiversity and heavy metal tolerance of endophytic fungifrom six dominant plant species in a Pb-Zn mine wasteland inChinardquo Fungal Ecology vol 5 no 3 pp 309ndash315 2012

[6] A E Arnold ldquoUnderstanding the diversity of foliar endo-phytic fungi progress challenges and frontiersrdquo Fungal BiologyReviews vol 21 no 2-3 pp 51ndash66 2007

[7] K D Hyde and K Soytong ldquoThe fungal endophyte dilemmardquoFungal Diversity vol 33 pp 163ndash173 2008

[8] G Strobel and B Daisy ldquoBioprospecting for microbial endo-phytes and their natural productsrdquoMicrobiology and MolecularBiology Reviews vol 67 no 4 pp 491ndash502 2003

[9] A E Arnold and F Lutzoni ldquoDiversity and host range of foliarfungal endophytes are tropical leaves biodiversity hotspotsrdquoEcology vol 88 no 3 pp 541ndash549 2007

[10] T S Suryanarayanan T S Murali N Thirunavukkarasu M BGovinda Rajulu G Venkatesan and R Sukumar ldquoEndophyticfungal communities in woody perennials of three tropical foresttypes of the Western Ghats southern Indiardquo Biodiversity andConservation vol 20 no 5 pp 913ndash928 2011

[11] R A Mittermeier N Myers P R Gil and C G MittermeierHotspots Earthrsquos Biologically Richest and Most Endangered Ter-restrial Ecoregions Cemex Conservation International Wash-ington DC USA 2000

[12] R D Kshirsagar and N P Singh ldquoSome less known eth-nomedicinal uses from Mysore and Coorg districts Karnatakastate Indiardquo Journal of Ethnopharmacology vol 75 no 2-3 pp231ndash238 2001

[13] N YoganarasimhanMedicinal Plants of India Karnataka Inter-line Publishing Bangalore India 1996

[14] B Schulz U Wanke S Draeger and H J Aust ldquoEndophytesfrom herbaceous plants and shrubs effectiveness of surfacesterilization methodsrdquo Mycological Research vol 97 no 12 pp1447ndash1450 1993

[15] H Barnett and B Hunter Illustrated Genera of Imperfect FungiBurgess Publishing Minneapolis Minn USA 1998

[16] K H DomschW Gams and T AndersonCompendium of SoilFungi Academic Press New York NY USA 2003

[17] J F Leslie and B A Summerell The Fusarium LaboratoryManual Blackwell Publishing London UK 2006

[18] W Photita P W J Taylor R Ford K D Hyde and S Lumy-ong ldquoMorphological and molecular characterization of Col-letotrichum species from herbaceous plants inThailandrdquo FungalDiversity vol 18 pp 117ndash133 2005

[19] V Kumaresan and T S Suryanarayanan ldquoOccurrence and dis-tribution of endophytic fungi in a mangrove communityrdquoMycological Research vol 105 no 11 pp 1388ndash1391 2001

[20] V Kumaresan and T S Suryanarayanan ldquoEndophyte assem-blages in young mature and senescent leaves of Rhizophora

apiculata evidence for the role of endophytes inmangrove litterdegradationrdquo Fungal Diversity vol 9 pp 81ndash91 2002

[21] J A Ludwig and J F Reynolds Statistical Ecology A Primer onMethods and Computing John Wiley amp Sons New York NYUSA 1988

[22] M Nei ldquoGenetic distance between populationsrdquo AmericanNaturalist vol 106 no 949 pp 283ndash293 1972

[23] M P Miller Tools For Population Genetic Analyses (TFPGA)V L3 A Windows program For the Analysis of Allozyme AndmoleculAr Genetic data Department of Biological SciencesNorthern Arizona University Flagstaff Ariz USA 1997

[24] D C Lacap K D Hyde and E C Y Liew ldquoAn evaluationof the fungal rsquomorphotypersquo concept based on ribosomal DNAsequencesrdquo Fungal Diversity vol 12 pp 53ndash66 2003

[25] W Y Huang Y Z Cai K D Hyde H Corke and M SunldquoBiodiversity of endophytic fungi associated with 29 traditionalChinese medicinal plantsrdquo Fungal Diversity vol 33 pp 61ndash752008

[26] M V Tejesvi B Mahesh M S Nalini et al ldquoFungal endophyteassemblages from ethnopharmaceutically important medicinaltreesrdquo Canadian Journal of Microbiology vol 52 no 5 pp 427ndash435 2006

[27] N Raj Coelomycetous Anamorphs With Appendage BearingConidia Edwards Brothers Ann Harbor Michigan 1993

[28] G A Strobel ldquoMicrobial gifts from rain forestsrdquo CanadianJournal of Plant Pathology vol 24 no 1 pp 14ndash20 2002

[29] M V Tejesvi B Mahesh M S Nalini et al ldquoEndophytic fungalassemblages from inner bark and twig of Terminalia arjunaW amp A (Combretaceae)rdquo World Journal of Microbiology andBiotechnology vol 21 no 8-9 pp 1535ndash1540 2005

[30] B Mahesh M V Tejesvi M S Nalini et al ldquoEndophyticmycoflora of inner bark of Azadirachta indica A Jussrdquo CurrentScience vol 88 no 2 pp 218ndash219 2005

[31] K Rajagopal S Kalavathy S Kokila et al ldquoDiversity of fungalendophytes in few medicinal herbs of southern Indiardquo AsianJournal of Experimental Biological Sciences vol 1 pp 415ndash4182010

[32] Y L Krishnamurthy S B Naik and S Jayaram ldquoFungal com-munities in herbaceous medicinal plants from the malnadregion Southern IndiardquoMicrobes and Environments vol 23 no1 pp 24ndash28 2008

[33] D L Hawksworth and A Y Rossman ldquoWhere are all theundescribed fungirdquo Phytopathology vol 87 no 9 pp 888ndash8911997

[34] D Wilson and G C CarrolL ldquoInfection studies of Disculaquercina an endophyte of Quercus garryanardquo Mycologia vol86 no 5 pp 635ndash647 1994

[35] G A Strobel W M Hess J-Y Li et al ldquoPestalotiopsis guepiniia taxol-producing endophyte of the wollemi pine Wollemianobilisrdquo Australian Journal of Botany vol 45 no 6 pp 1073ndash1082 1997

[36] K Clay ldquoFungal endophytes of grassesrdquo Annual Review ofEcology and Systematics vol 21 no 1 pp 275ndash297 1990

[37] P Bayman L L Lebron R L Tremblay and D J Lodge ldquoVari-ation in endophytic fungi from roots and leaves of Lepanthes(Orchidaceae)rdquoNewPhytologist vol 135 no 1 pp 143ndash149 1997

[38] A EArnold ZMaynard andG SGilbert ldquoFungal endophytesin dicotyledonous neotropical trees patterns of abundance and

International Journal of Biodiversity 9

diversityrdquo Mycological Research vol 105 no 12 pp 1502ndash15072001

[39] P F Cannon andCM Simmons ldquoDiversity andhost preferenceof leaf endophytic fungi in the Iwokrama Forest ReserveGuyanardquoMycologia vol 94 no 2 pp 210ndash220 2002

[40] X Sun Q Ding K D Hyde and L D Guo ldquoCommunitystructure and preference of endophytic fungi of three woodyplants in a mixed forestrdquo Fungal Ecology vol 5 no 5 pp 624ndash632 2012

[41] P HollidayADictionary of Plant Pathology CambridgeUniver-sity Press Cambridge UK 1998

[42] J Collado G Platas and F Pelaez ldquoIdentification of an endo-phytic Nodulisporium sp from Quercus ilex in central Spainas the anamorph of Biscogniauxia mediterranea by rDNAsequence analysis and effect of different ecological factors ondistribution of the fungusrdquo Mycologia vol 93 no 5 pp 875ndash886 2001

[43] X-X Gao H Zhou D-Y Xu C-H Yu Y-Q Chen and L-HQu ldquoHigh diversity of endophytic fungi from the pharmaceuti-cal plant Heterosmilax japonica Kunth revealed by cultivation-independent approachrdquo FEMS Microbiology Letters vol 249no 2 pp 255ndash266 2005

[44] N S Raviraja ldquoFungal endophytes in five medicinal plantspecies from Kudremukh Range Western Ghats of IndiardquoJournal of Basic Microbiology vol 45 no 3 pp 230ndash235 2005

[45] A E Arnold Z Maynard G S Gilbert P D Coley and T AKursar ldquoAre tropical fungal endophytes hyperdiverserdquo EcologyLetters vol 3 no 4 pp 267ndash274 2000

[46] B S Naik J Shashikala and Y L Krishnamurthy ldquoDiversityof fungal endophytes in shrubby medicinal plants of Malnadregion Western Ghats Southern Indiardquo Fungal Ecology vol 1no 2-3 pp 89ndash93 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

International Journal of Biodiversity 9

diversityrdquo Mycological Research vol 105 no 12 pp 1502ndash15072001

[39] P F Cannon andCM Simmons ldquoDiversity andhost preferenceof leaf endophytic fungi in the Iwokrama Forest ReserveGuyanardquoMycologia vol 94 no 2 pp 210ndash220 2002

[40] X Sun Q Ding K D Hyde and L D Guo ldquoCommunitystructure and preference of endophytic fungi of three woodyplants in a mixed forestrdquo Fungal Ecology vol 5 no 5 pp 624ndash632 2012

[41] P HollidayADictionary of Plant Pathology CambridgeUniver-sity Press Cambridge UK 1998

[42] J Collado G Platas and F Pelaez ldquoIdentification of an endo-phytic Nodulisporium sp from Quercus ilex in central Spainas the anamorph of Biscogniauxia mediterranea by rDNAsequence analysis and effect of different ecological factors ondistribution of the fungusrdquo Mycologia vol 93 no 5 pp 875ndash886 2001

[43] X-X Gao H Zhou D-Y Xu C-H Yu Y-Q Chen and L-HQu ldquoHigh diversity of endophytic fungi from the pharmaceuti-cal plant Heterosmilax japonica Kunth revealed by cultivation-independent approachrdquo FEMS Microbiology Letters vol 249no 2 pp 255ndash266 2005

[44] N S Raviraja ldquoFungal endophytes in five medicinal plantspecies from Kudremukh Range Western Ghats of IndiardquoJournal of Basic Microbiology vol 45 no 3 pp 230ndash235 2005

[45] A E Arnold Z Maynard G S Gilbert P D Coley and T AKursar ldquoAre tropical fungal endophytes hyperdiverserdquo EcologyLetters vol 3 no 4 pp 267ndash274 2000

[46] B S Naik J Shashikala and Y L Krishnamurthy ldquoDiversityof fungal endophytes in shrubby medicinal plants of Malnadregion Western Ghats Southern Indiardquo Fungal Ecology vol 1no 2-3 pp 89ndash93 2008

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology

Submit your manuscripts athttpwwwhindawicom

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Anatomy Research International

PeptidesInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporation httpwwwhindawicom

International Journal of

Volume 2014

Zoology

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Molecular Biology International

GenomicsInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

The Scientific World JournalHindawi Publishing Corporation httpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioinformaticsAdvances in

Marine BiologyJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Signal TransductionJournal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

BioMed Research International

Evolutionary BiologyInternational Journal of

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Biochemistry Research International

ArchaeaHindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Genetics Research International

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Advances in

Virolog y

Hindawi Publishing Corporationhttpwwwhindawicom

Nucleic AcidsJournal of

Volume 2014

Stem CellsInternational

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

Enzyme Research

Hindawi Publishing Corporationhttpwwwhindawicom Volume 2014

International Journal of

Microbiology