ENVIRONMENTAL MICROBIOLOGY

Season and Tissue Type Affect Fungal EndophyteCommunities of the Indian Medicinal Plant Tinosporacordifolia More Strongly than Geographic Location

Ashish Mishra & Surendra K. Gond & Anuj Kumar &

Vijay K. Sharma & Satish K. Verma &

Ravindra N. Kharwar & Thomas N. Sieber

Received: 25 June 2011 /Accepted: 15 February 2012 /Published online: 20 March 2012# Springer Science+Business Media, LLC 2012

Abstract A total of 1,151 endophytic fungal isolates repre-senting 29 taxa were isolated from symptom-less, surface-sterilized segments of stem, leaf, petiole, and root of Tino-spora cordifolia which had been collected at three locationsdiffering in air pollution in India (Ramnagar, Banaras HinduUniversity, Maruadih) during three seasons (summer, mon-soon, winter). Endophytes were most abundant in leaf tis-sues (29.38% of all isolates), followed by stem (18.16%),petiole (10.11%), and root segments (6.27%). The frequencyof colonization (CF) varied more strongly among tissue typeand season than location. CF was maximal during monsoonfollowed by winter and minimal during summer. A specieseach of Guignardia and Acremonium could only be isolatedfrom leaves, whereas all other species occurred in at leasttwo tissue types. Penicillium spp. were dominant (12.62%of all isolates), followed by Colletotrichum spp. (11.8%),Cladosporium spp. (8.9%), Chaetomium globosum (8.1%),Curvularia spp. (7.6%), and Alternaria alternata (6.8%).Species richness, evenness, and the Shannon–Wiener diver-sity index followed the same pattern as the CF with thetissue type and the season having the greatest effect on these

indices, suggesting that tissue type and season are moreinfluential than geography. Dissimilarity of endophyte com-munities in regards to species composition was highestamong seasons. Colletotrichum linicola occurred almostexclusively in winter, Fusarium oxysporum only in winterand summer but never during monsoon and Curvularialunata only in winter and during monsoon but never insummer. Emissions of NO2, SO2, and suspended particulatematter were negatively correlated with the CF. Ozone didnot have any effect. The frequency of most species declinedwith increasing pollution, but some showed an oppositetrend (e.g., Aspergillus flavus). Five unnamed taxa (sterilemycelia) were identified as Aspergillus tubingensis, Colle-totrichum crassipes, Botryosphaeria rhodina, Aspergillussydowii, and Pseudofusicoccum violaceum, using moleculartools. Fifteen of the 29 endophyte taxa exhibited antibacte-rial activity. B. rhodina (JQ031157) and C. globosumshowed activity against all bacterial human pathogens test-ed, with the former showing higher activity than the latter.

AbbreviationsT, L, P, and R assignment code for stem, leaf, petiole,

and root tissuesW, S, M for winter, summer, and monsoonLoc for locationMMTL for Mycopathology and Microbial

Technology Laboratory

Introduction

Endophytes are hidden and largely unexplored entities ofthe microbial world. They colonize inter- or intracellular

A. Mishra : S. K. Gond :V. K. Sharma : S. K. Verma :R. N. Kharwar (*)Mycopathology and Microbial Technology Laboratory,Department of Botany, Banaras Hindu University,Varanasi 221005, Indiae-mail: rnkharwar@yahoo.com

A. KumarDepartment of Botany, Budhha P. G. College,Kushinagar, India

T. N. SieberETH Zurich, Institute of Integrative Biology,Forest Pathology and Dendrology,8092, Zurich, Switzerland

Microb Ecol (2012) 64:388–398DOI 10.1007/s00248-012-0029-7

spaces of healthy tissues of most plants studied to date. DeBary [14] introduced the term “endophyte” for organismsliving inside healthy tissues. Bacon et al. [4] slightly ex-panded De Bary’s (1866) definition by defining endophyteas “microbes that colonize living, internal tissues of plantswithout causing any immediate, and overt negative symp-toms”. Endophytes range from fungi [19, 44, 48, 71] tobacteria [24] including actinomycetes [64], but most studiedendophytes are fungi. Some endophytes are considered tomediate interactions between plants and their competitors,seed dispersers, herbivores, and pathogens [2, 10, 20, 55].However, biology and ecology of most endophytes are notknown but are assumed to vary according to host andenvironment [7, 8, 38]. A huge number of authors suggestedthat endophytes are good sources of novel secondary metab-olites that possess antibacterial [37, 65] antifungal [15, 65]and anticancer properties [36]. In this study, the endophyticmycobiota of the medicinal plant Tinospora cordifoliaMiers., was studied. T. cordifolia, commonly known asGuduchi, Gurch, Giloe or Amrita, is a large, glabrous,deciduous, shade-loving climbing shrub belonging tothe family of the Menispermiaceae. This plant is nativeto India, and distributed throughout the tropical Indiansubcontinent, ascending to an altitude of 300 m. Differ-ent classes of compounds such as alkaloids (berberine,protoberberine, palmatine, etc.), glycosides (tinocordi-side, cordifolioside A and B), diterpenoids lactones (fur-anolactone, tinosorides, jateorine), steroids (β-sitosterol,giloinsterol), sesquiterpenoid (Tinocordifolin), and ali-phatic compounds (octacosanol, heptacosanol, andnonacosan-15-one) have been isolated from differentparts of T. cordifolia [57].

Secondary metabolites extracted from plants are usuallyconsidered to have been produced by the plant. However,some of these metabolites may in fact have been producedby endophytic fungal colonizers in the plant. This is the casefor fungal taxol, a potent antibreast cancer drug, beingproduced by the endophytic fungus Taxomyces andreanaein Taxus brevifolia [58]. Thus, to learn if any endophyticmicrobe may be producing one or more of a plethora ofbioactive substances known from T. cordifolia, it would firstbe critical to do a systematic study on the endophytes of thisplant and to know their biology and distribution both withinthe plant and in plants from several locations. Therefore,species richness, diversity, and colonization frequency ofendophytic fungi were examined in four different planttissues collected at three locations differing in emissions ofair pollutants during three seasons to evaluate tissue speci-ficity of endophytes and the influence of season and airpollution on the community of endophytic mycobiota. Pre-liminary tests for antibacterial activity were performed withcrude extracts of representative taxa. A collection of suchendophytes could then serve as a library to begin more

comprehensive fermentation, screening, and chemical char-acterization studies.

Materials and Methods

Sample Collection from Selected Sites

Mature, healthy, symptomless succulent stems (T), leaves(L), petioles (P), and soil roots (R) were collected from threeindividual plants of T. cordifolia at each of three locationswith similar climatic conditions: average amount of rainfallis 5 mm in winter , 50 mm in summer, and 1,010 mm duringmonsoon; mean temperature is 20°C in winter, 30°C insummer, and 28°C during monsoon; relative air humidityis 65% in winter, 45% in summer, and 85% during mon-soon. However, the locations strongly differed in regards tothe emissions of air pollutants (Table 1). Three stems (length,6–8 cm; diameter, 4–8 mm), three leaves having surface areasof between 30 and 105 cm2, three petioles (length, 6–7 cm;diameter, 2–4 mm), and three root segments (length, 6–8 cm;diameter, 3–8 mm) were collected per plant in each of thethree seasons winter, monsoon, and summer between Novem-ber 2007 and September 2008. Samples were collected fromthe same plants in each season. T. cordifolia is a vine. All theplants examined in this study used mango trees (Mangiferaindica) as support. All samples except roots were taken at aheight of at least 1.5 m above ground level and put separatelyin sterile polybags, brought to the laboratory in an ice box,stored at 4°C and processed within 48 h.

Plant Surface—Sterilization, Isolation, and Identification

Samples were washed thoroughly in running tap water,rinsed with double-distilled water, and surface-sterilizedaccording to Petrini [45]. Samples were dipped in 70%ethanol for 1 min, immersed in aqueous solution of NaOCl(4% available chlorine) for 3 min followed by immersion in70% ethanol for 10 s. The samples were then rinsed indouble-distilled sterile water and dried under aseptic condi-tion. Each leaf, stem, root, and petiole was cut into 22 or 23

Table 1 Site characteristics

Air pollutants (μg m−3)

Site Name Coordinates SO2 NO2 O3 SPM

Loc1 BHU 25° 16′ 05.36″ N 12 20 50 16582° 59′ 20.51″ E

Loc2 Maruadih 25° 17′ 56.09″ N 81.3 87.4 41 58982° 58′ 22.12″ E

Loc3 Ramnagar 25° 16′ 03.55″ N 39 49 29 44083° 01′ 30.40″ E

Endomycoflora of Tinospora cordifolia 389

segments (200 segments per season, location and tissuetype, i.e., 66 or 67 segments per tissue type and plant),measuring approximately 0.5×0.5 cm for leaves or 0.5 cmin length for stems, petioles, roots (these three tissues hadbeen split axially once prior to segmentation), and placed onPetri dishes containing potato dextrose agar (PDA) supple-mented with streptomycin (200 mgl−1) and incubated at 26±1°C (Caltan BOD Incubator-152, Narang Scientific Works,New Delhi, India). Four different media had initially beentested (malt extract agar, mycological agar, nutrient agar,and PDA from HiMedia Laboratories Pvt. Ltd, India), butPDA was found to give the best results. Per tissue andseason, 600 segments were examined amounting to 7,200tissue segments during the whole study. The tissues werechecked every other day for 21 days, and actively growingfungal mycelia transferred to new PDA plates for purifica-tion and identification. The method of Schulz et al. [54] wasapplied to check the effectiveness of surface sterilization.The fungi were identified to the genus and/or species levelbased on colony morphology and micromorphology (conid-ia, conidiophores, and fruit body morphology) using stan-dard fungal taxonomic manuals [1, 6, 16, 49, 67]. Allisolates received a specific code number (MMTL 2108-3259) and were deposited at the department of Botany,Banaras Hindu University, Varanasi, India in lyophilizedform in separate cryovials at −20°C (Blue Star).

Molecular Characterization of Unidentified Endophytic Fungi

Genomic DNA was extracted and amplified from myceliasterilia following the slightly modified protocol of Sim et al.[56]. The universal primers ITS1 5′ TCCGTAGGT-GAACCTGCGG 3′ and ITS4 5′ TCCTCCGCTTATTGA-TATGC 3′ (Metabion International, Martinsried, Germany)were used to amplify the 5.8S rDNA and two internaltranscribed spacer (ITS) regions flanked by the 18S and28S rRNA genes. Total PCR mixture of 25 μl, each con-taining 1 μl (100 ng/μl) of DNA template, 1 μl of eachprimer, 0.33 μl (3 unit) Taq polymerase, 1.5 μl MgCl2,0.25 μl DNTPs, buffer (10×) 2.5 μl and 17.42 μl MQ waterfor each reaction mixture. The PCR reactions were per-formed in icycler (BioRad) with the following conditions:predenaturation at 94°C for 4 min, 35 cycles at 94°C (dena-turation) for 1 min, 55°C (annealing) for 1 min, 72°C(extension) for 1 min and then a final extension for 5 minat 72°C. Amplified PCR products were resolved by electro-phoresis in 1.5% (w/v) agarose gels stained with ethidiumbromide (0.5 μg/ml) for visual examination. PCR productwas sent to First BASE Laboratories (Malaysia) for se-quencing. Obtained ITS rDNA sequences were comparedby Basic Local Alignment Search Tool search among men-tion sequences at the National Center for BiotechnologyInformation GenBank for the identification of sequences.

Statistical Analysis

The frequency of colonization (CF) was expressed in per-centages and calculated as the number of segments colo-nized by a single endophyte divided by the total number ofsegments examined ×100 [26]. Effects of location, tissuetype, and season on CF were examined using analysis ofvariance (ANOVA) and displayed as boxplots [12]. Speciesrichness, i.e., the number of species, Shannon–Wiener index[41] and Whittaker’s evenness [70] were calculated to ex-press diversity of endophytes in stem, leaf, petiole, and rootat the three locations during the three seasons. The threediversity indices were subjected to ANOVA to determineeffects of location, tissue type, and season. Jaccard’s dis-tance [33] was determined between each of the two sam-pling units. The resulting matrix was subjected to clusteranalysis using the Ward algorithm to visualize similarity oflocations, seasons, and tissue types in regards to speciescomposition of the endophyte community. Seasonal effectson the frequency of the 14 most frequent endophytes weredisplayed as biplots. Regression analysis was used to iden-tify effects of air pollutants on the colonization by endo-phytes. The software “R” was used for all the statisticalanalyses [47].

Fermentation and Extraction of Metabolite

The endophytes were transferred to fresh PDA platesand allowed to grow at 26°C±1°C for 7–14 days. Somecolonized plugs of PDA (5 mm in diameter) weretransferred into 2,000 ml Erlenmeyer flask containing1,000 ml potato dextrose broth. Flasks were put on ashaker in an incubator (orbital shaking incubator Remi-RIS-24BL) at 120 rpm for 14–21 days at 26°C±1°C.Metabolites were extracted thrice with ethyl acetate(with equal volume) at room temperature and concen-trated in a rotary vacuum evaporator to get the residuedry (crude) prior to antimicrobial assays.

Antimicrobial Bioassay

The paper disk diffusion bioassay was done adoptingthe methodology by Hadacek and Greger [23]. Thecrude compound was dissolved in methanol to make afinal concentration of 5 mg/ml. Twenty microliters wereapplied onto each 5-mm diameter paper disk. Afterevaporation of the organic solvent, the disks wereplaced to the center of 9 cm diameter Muller Hintonagar plates previously inoculated with 0.5 ml sporessuspension (104 CFU/ml) of different human bacterialpathogens. After 3 days, the widths of the inhibitionzones were measured (in millimeter). All experimentswere done in three replicates.

390 A. Mishra et al.

Results

A total of 1,151 (CF016%) endophytic fungal isolates weresuccessfully isolated from 7,200 tissue segments, represent-ing 29 endophytic fungal species. Five of 29 endophytictaxa (five sterile mycelia) were morphologically unidentifi-able and therefore, subjected to molecular identification.Three sterile mycelia no. MMTL 3130 (NCBI genbankaccession no. JQ031155), MMTL 3150 (accession no.JQ031156) and MMTL 3177 (accession no. JQ031157)showed 99% sequence similarity with Aspergillus tubingen-sis, Colletotrichum crassipes, and Botryosphaeria rhodina,while the two sterile mycelia no. MMTL3215 (accession no.JQ031158) and MMTL 3251 (accession no. JQ031159)showed 97% and 98% sequence similarity with Aspergillussydowii and Pseudofusicoccum violaceum, respectively.Most isolates produced conidia in culture (81.57%), where-as ascospores developed in 11.3% of the isolates (Table 2).The tissue type (p<0.0001) and the season (p00.0004) hada significant influence on the colonization by endophytesbut not the location (p00.95; Fig. 1). Endophytes were mostabundant in leaf tissues (29.38% of the isolates), followedby stem (18.16%), petiole (10.11%) and root segments(6.27%). However, CF varied strongly within tissue typesaccording to the season. CF was maximal during monsoon(23.23%) followed by winter (15.35%) and minimal duringsummer (8.85%). CF was low for all species and laid be-tween 0.5% and 4% for most species. A maximum CF of9.5% was observed for Colletotrichum linicola in leaves.All the 29 detected species could be isolated from leaves, 26from stems, 23 from petioles, and 18 from roots. Sixteenspecies could be detected in all tissue types. Guignardia sp.and Acremonium sp. could only be isolated from leaves,whereas all other species occurred in at least two tissuetypes. Among the isolates, Penicillium spp. were dominant(12.62% of all isolates), followed by Colletotrichum spp.(11.75%), Cladosporium spp. (8.93%), Chaetomium globo-sum (8.06%), Curvularia spp. (7.55%) and Alternaria alter-nata (6.75%). Trichoderma viride,Monilia sp., Acremoniumsp., and Guignardia sp. were rare (0.69%, 0.86%, 0.52%and 0.52%; Table 2). Species richness, evenness, and theShannon–Wiener diversity index followed the same patternas the CF with the tissue type (p≤0.001) and the season (p≤0.006) having the greatest effect on these indices, suggest-ing that tissue type and season are more influential than thegeographic location (Fig. 2). Dissimilarity of endophytecommunities in regards to species composition as expressedby the Jaccard’s distance (JC) was highest among seasons(Fig. 3). Some species of endophytes occurred preferentiallyin one or two of the seasons. For example, C. linicolaoccurred almost exclusively in winter and Fusarium oxy-sporum only in winter and summer but never during mon-soon (Fig. 4, Table 2). In contrast, Curvularia lunata was

found only in winter and during monsoon but never insummer. Although the effect of season and tissue type onCF and species diversity was much more pronouncedthan the effect of the location, emissions of NO2

(p00.0177), SO2 (p00.0153), and suspended particulatematter (SPM; p00.0456) were negatively correlated withthe CF (Fig. 5). Ozone did not have any effect (p00.816). Frequency of most species declined with increas-ing pollution, but some showed an opposite trend asAspergillus flavus (Fig. 5). Fifteen (51.72%) of 29 endo-phyte taxa exhibited antibacterial activity against at leastone of eight bacterial human pathogens. B. rhodina (JQ031157 “MMTL-3177”) and C. globosum were the mostactive taxa and inhibited all pathogens tested followed byF. oxysporum, Colletotrichum dematium, and Penicilliumsp. 1 and 2 (Table 3).

Discussion

Diversity and frequency of endophytic fungi were primarilyinfluenced by tissue type and season (Figs. 1 and 2). Fre-quency of colonization and species richness were highest inthe leaves and lowest in the roots. This may be due to theleaves attaining highest canopy cover compared to othertissues, providing greater surface area for inoculum capture[9]. In addition, the leaves of T. cordifolia also have lessantimicrobial property than the stem and root [61] whichperhaps promotes the endophytic colonization of this organ.The result confirms earlier studies about fungal endophytefrequency and diversity of other important medicinal plantsin India [48, 50, 63]. The roots were least colonized prob-ably because jatrorrhizine, an antimicrobial metabolitefound in root of T. cordifolia, may have suppressed growthof some endophytes [57]. The environmental conditions inthe rhizosphere are much more uniform and stable thanthose above the ground, and this uniformity may be respon-sible for high evenness and low species richness and diver-sity observed in roots. In addition, airborne spores fromdistant sources can colonize leaves whereas roots get mainlycolonized by inoculum present in the nearby soil. However,in the present study, only a species each of Guignardia andAcremonium appeared to occur specifically in leaves whileall other species were detected in at least two of the exam-ined tissues. Similarly, Guignardia mangiferae showedspecificity for leaves of the medicinal orchid Dendrobiumnobile [72]. C. globosum was reported to be confined tobark [19] or leaf [38] in earlier studies, but occurred in everytissue in this study. Endophytic fungi often are tissue spe-cific, but most show “only” tissue preference [13, 30]. Forexample, none of the nonxylariaceous species isolated fromeither leaf or petiole tissue of the palm Trachycarpus for-tunei was tissue specific [59]

Endomycoflora of Tinospora cordifolia 391

Tab

le2

Seasonalvariationof

endo

phytic

recovery

(%CF)from

differentlocatio

nsandtissuetypes

Endophytic

fungi

Winter

Sum

mer

Monsoon

LOC1

LOC2

LOC3

LOC1

LOC2

LOC3

LOC1

LOC2

LOC3

TL

PR

TL

PR

TL

PR

TL

PR

TL

PR

TL

PR

TL

PR

TL

PR

TL

PR

Total

CF%

sp.1

1.5

1.0

3.0

2.5

0.5

1.5

2.0

0.5

0.5

2.0

1.5

2.0

1.0

1.5

1.0

1.5

1.0

1.0

2.0

2.0

1.5

3.0

1.0

3.5

6.0

2.0

1.29

sp.2

1.5

0.5

0.5

0.5

3.5

1.5

2.0

2.0

0.5

2.0

1.0

0.43

sp.3

1.0

2.0

0.08

sp.4

1.0

2.0

0.5

1.0

1.0

1.0

1.5

4.0

3.0

1.0

0.5

1.0

0.5

4.0

3.5

1.5

1.0

1.5

1.0

4.5

3.0

0.5

1.06

sp.5

4.5

9.5

1.0

0.5

2.0

9.0

0.5

1.0

1.0

0.80

sp.6

3.0

0.08

sp.7

1.0

2.0

1.0

0.5

0.5

1.5

2.5

1.0

1.0

1.5

0.5

2.0

1.5

0.5

2.0

2.0

1.0

4.0

0.5

0.5

2.5

1.0

2.0

1.0

3.5

2.0

1.08

sp.8

1.0

2.5

1.0

1.0

1.5

2.5

1.5

3.0

2.0

3.0

3.5

2.5

1.0

0.72

sp.9

0.5

1.5

1.5

1.5

1.0

2.5

0.5

1.0

2.0

1.0

2.0

1.5

1.0

1.5

2.0

1.5

1.0

1.5

3.5

1.0

1.0

0.84

sp.10

1.0

1.0

0.5

2.5

0.5

1.5

1.0

1.0

1.5

1.0

2.5

0.38

sp.11

1.0

0.5

0.5

1.5

2.0

0.5

0.5

1.5

0.22

sp12

2.0

2.5

0.5

1.0

0.5

0.5

1.0

0.22

sp.13

0.5

2.5

0.5

3.0

1.5

2.0

1.5

0.5

1.5

1.5

1.5

2.5

1.0

1.5

3.5

0.5

1.5

3.5

2.0

0.5

1.0

3.0

3.5

3.0

1.20

sp.14

3.5

4.5

2.0

1.5

2.5

1.5

0.43

sp15

1.5

1.0

2.0

2.0

2.0

1.5

1.5

1.0

1.0

2.0

3.0

1.0

3.5

5.0

0.77

sp.16

3.5

1.5

1.0

3.5

3.0

0.34

sp.17

1.5

1.0

1.5

1.5

2.0

6.5

0.5

1.0

2.0

0.48

sp.18

1.5

2.5

1.5

1.0

1.5

1.0

0.5

2.0

0.31

sp.19

2.5

1.5

1.0

0.13

sp.20

1.0

2.0

1.0

0.5

2.5

1.0

0.5

1.5

2.0

2.0

0.5

2.5

1.5

1.0

3.0

1.0

1.5

2.0

0.5

1.5

4.0

2.0

0.97

sp.21

0.5

0.5

0.5

0.5

2.0

0.5

2.5

2.0

0.5

2.0

3.0

2.5

2.0

1.0

1.5

2.0

1.5

4.5

1.0

0.5

1.5

1.5

1.5

5.5

1.5

1.0

1.20

sp.22

1.5

0.5

4.5

2.0

2.0

3.0

3.0

1.0

1.5

3.0

0.5

1.0

3.0

0.5

2.0

0.80

sp.23

1.0

0.5

1.5

1.0

0.11

sp.24

3.5

1.0

0.5

0.13

sp.25

1.0

2.0

1.5

2.0

1.0

2.0

0.5

0.27

sp.26

5.0

2.0

1.0

1.5

1.5

2.5

0.37

sp.27

1.0

3.5

0.5

1.0

1.0

1.0

1.0

0.5

0.5

1.0

0.5

2.0

2.0

3.5

0.52

sp.28

0.5

1.5

1.0

1.0

1.0

0.5

1.5

0.5

1.0

4.5

1.0

1.0

2.0

1.0

0.50

sp.29

1.5

0.5

1.0

0.5

0.5

0.11

totalCf%

0.36

0.97

0.27

0.18

0.33

0.41

0.23

0.18

0.52

1.12

0.25

0.19

0.37

0.45

0.18

0.09

0.20

0.29

0.15

0.04

0.34

0.44

0.22

0.06

0.86

1.51

0.41

0.29

0.40

0.45

0.23

0.22

0.91

1.55

0.52

0.29

sp.1,C.g

lobo

sum;sp.2,E

mericella

nidu

lans;sp.3,Guign

ardiasp.;sp.4,C.d

ematium;sp.5,C.lin

icola;

sp.6,A

crem

onium

sp.;sp.7,A.a

lternata;

sp.8,A

.flavus;sp.9,A

.niger;sp.10,

A.terreus;

sp.11,

Botrytis

sp.;sp.1

2,Clado

sporiumap

icale;sp.13,

C.clado

sporioides;sp.14

,Curvulariainterm

edia;sp.15

,C.lun

ata;

sp.16,

Drechsleragram

inea;sp.17

,F.o

xysporum

;sp.18

,Hum

icolasp.;

sp.19,

Mon

iliasp.;sp.20,

Nigrosporaoryzae;sp.21,

Penicillium

sp.1;sp.22,

Penicillium

sp.2;sp.23,

T.viride;sp.24,Verona

eamusae;sp.25

,A.tubing

ensis(JQ03

1155

),26,C.crassipes

(JQ03

1156

);27

,B.rhod

ina(JQ03

1157

);28

,A.sydo

wii(JQ03

1158

);29

,P.

violaceum

(JQ03

1159

).Tstem

,Lleaf,Ppetio

le,Rroot

392 A. Mishra et al.

Many workers found that the level of endophytic coloni-zation increased during rainy seasons [48, 51, 71] and this isin accordance with our results. For example, Tejesvi et al.[60] found only five species in winter compared to 19species during monsoon in the bark of Terminalia arjuna[66]. However, there are also contrasting reports about

higher frequencies of colonization in other seasons, e.g.,Naik et al. [43] obtained significantly more isolates duringthe winter season than the monsoon or summer season fromshrubby medicinal plants collected in Southern India.Higher humidity and temperature during monsoon mayfavor a majority of endophytes but not all. For example,C. linicola, which causes anthracnose on flax (Linum usita-tissimum) [34], has not been detected as an endophyte in anyother plant species, so far, but was present quite frequentlyand almost only in winter in leaves of T. cordifolia atlocations 1 and 3 (Fig. 4). These locations are situated in aregion with high production of flax. Thus, a “jump” of C.linicola from L. usitatissimum to T. cordifolia is conceiv-able. The main source of infection are plant residues onwhich the fungus sporulates preferentially when air humid-ity is high and rainfall rare which is the case in the studyarea during winter. A similar mode of life may apply to F.oxysporum of which more than 100 formae speciales fromvarious hosts are known [21] including flax on which it wasdescribed to cause wilting [22]. C. lunata was found only inwinter and during monsoon but never in summer (Fig. 4). Itwas also one of the dominant endophytes in leaves ofBauhinia phoenicea and several species of ethnopharma-ceutically important medicinal herbs in India [39, 50].

Geography affected patterns of distribution of endo-phytes more strongly than the season in many studies [11],but our results clearly indicate the season’s dominance overlocation (Fig. 3), although the three locations differed sig-nificantly in regards to air pollution (Table 1). Nevertheless,emissions of NO2, SO2, and SPM were negatively correlatedwith the CF, and, thus, the CF was lowest at the highlypolluted location 2. The frequency of most species declinedwith increasing pollution, but some showed an oppositetrend as A. flavus (Fig. 5). Decreasing frequencies of colo-nization by endophytes with increasing air pollution wasdemonstrated in several studies. Best studied in this regardare pine and birch species. Simulated acid rain (SAR) ad-justed to pH 3 significantly reduced density of colonizationof birch leaves by endophytes [27], and frequency of colo-nization of the twig-endophyte Ophiovalsa betulae de-creased with increasing concentration of ambient NO2 innatural stands of Betula pubescens [5]. Endophyte densitiesin B. pubescens and B. pendula were low near a copper–nickel smelter, and correlated negatively with foliar concen-trations of heavy metals and aerial SO2 levels [40]

The frequency of colonization of 2- to 3-year-old needlesof Japanese black pine by endophytic Lophodermium waslower in SAR-treated than control trees [3]. The total num-ber of needles infected with endophytes and also the numberof needles infected with one of the most frequent endo-phytes, Cenangium ferruginosum, were significantly lowernear heavy metals and sulfur-emmitting factories than in thecontrol area [28].

Figure 1 Boxplots depicting the effects of season and tissue type onthe variation of the frequency of colonization (percentage) by endo-phytic fungi

Figure 2 Boxplots depicting the effects of season and tissue type onthe variation of the species diversity (Shannon index) of the endophytecommunities

Endomycoflora of Tinospora cordifolia 393

Airborne fungi with high spore production, e.g., Penicil-lium spp., Cladosporium spp., or Alternaria spp., were themost frequent endophytic fungi in this study. Isolation ofsuch species always causes some uncertainty as to whethersurface sterilization has been strong enough in killing all the

epiphytic propagules. However, the surface sterilizationused was quite strong and results from the tests of effective-ness of surface sterilization were good [54]. Moreover, thesefungi often are among the most frequently isolated endo-phytes in many studies especially when plants in tropical

Figure 3 Dendrogramproduced using the Wardalgorhithm showing thesimilarity of the speciescomposition of the endophytecommunities of the samplingunits (W winter, M monsoon, Ssummer; 1–3 correspond to thelocation numbers (Table 1). Lleaf, T stem, P petiole, R root)

-0.4 -0.2 0.0 0.2 0.4 0.6

-0.4

-0.2

0.0

0.2

0.4

0.6

Principal Component 1

Prin

cipa

l Com

pone

nt 2

Chaetomium globosum

Colletotrichum dematium

Colletotrichum linicola

Alternaria alternataCladosporium cladosporioides

Curvularia lunata

Fusarium oxysporum

Nigrospora oryzae

Botryosphaeria rhodina

Aspergillus flavus

Aspergillus nigerPenicillium sp. 1

Penicillium sp. 2

Aspergillus sydowii

-0.1 0.0 0.1 0.2

-0.1

0.0

0.1

0.2

Winter

Summer

Monsoon

Figure 4 Biplot depicting the influence of the three seasons on themost frequently isolated endophyte species

Figure 5 Influence of NO2 emissions on the frequency of colonizationby endophytic fungi. Each symbol type represents one endophytespecies. The solid line shows the general trend and the broken linesdepict the effect of NO2 on two selected endophyte species.

394 A. Mishra et al.

regions were examined [18, 46, 62]. Thus, these fungirepresent important components in many endophytecommunities.

Species of Penicillium, Colletotrichum, Cladosporium,Chaetomium, Curvularia, and Alternaria were the dominantspecies in this study (Table 2), and it may be due to highspore production of these fungi and their cosmopolitannature, which statistically increases their chance to getestablished as endophytes [50, 53, 63]. The probability ofdetection of incidental species is usually proportional to thesampling size and method chosen, rather than the host itself[44]. Our results strongly indicate that the sampling size and

methods were adequate and thorough for rare speciesrecovery.

The level of resistance of pathogens against antibiotics isan increasing concern and, therefore, novel antimicrobialsare urgently needed. Endophytic fungi are considered po-tential sources of such metabolites after the discovery oftaxol from T. andreanae, an endophyte of T. brevifolia [58].More than 150 bioactive compounds were isolated during ascreening of 6,500 endophytic fungi, and more than 50% ofthese compounds were significantly active against variouspathogenic microbes [31]. More than half of the endophytetaxa showed antibacterial activity, corroborating earlier

Table 3 Antibacterial activity of crude extract of endophytic fungi isolated from T. cordifolia

Endophytic fungi Isolate no. Diameter of inhibition zone (mean±SE in mm) of human pathogenic bacteriaa (5 mg/ml)

A B C D E F G H

C. globosum MMTL 2108 10.00±0.57 11.66±0.33 11.66±0.33 13.66±0.33 17.50±0.28 13.00±0.57 15.00±0.57 19.33±0.33

Emericella nidulans MMTL 2201 0 0 0 0 0 0 0 0

Guignardia sp. MMTL 2232 0 11.00±0.57 0 0 0 10.00±0.00 0 0

C. dematium MMTL 2238 11.16±0.44 0 09.83±0.16 09.00±0.57 0 0 0 0

C. linicola MMTL 2315 12.00±0.00 0 0 0 0 0 0 0

Acremonium sp. MMTL 2373 0 0 0 0 0 0 0 0

A. alternata MMTL 2379 0 0 0 9.00±0.00 0 0 0 0

A. flavus MMTL 2457 11.33±0.66 0 0 9.33±0.33 0 0 0 0

A. niger MMTL 2509 0 0 0 0 0 0 0 0

A. terreus MMTL 2570 0 0 0 0 0 0 0 0

Botrytis sp. MMTL 2598 0 0 0 0 0 0 0 0

Cladosporium apicale MMTL 2614 0 0 0 0 0 0 0 0

C. cladosporioides MMTL 2630 10.00±0.00 0 0 0 0 0 0 0

Curvularia intermedia MMTL 2717 0 0 0 0 0 0 0 0

C. lunata MMTL 2748 24.6±0.33 0 0 0 0 0 0 15.50±0.50

Drechslera graminea MMTL 2804 0 0 0 0 0 0 0 12.30±0.66

F. oxysporum MMTL 2829 0 18.30±0.33 0 0 0 0 13.30±0.66 13.60±0.33

Humicola sp. MMTL 2864 0 0 0 0 0 0 0 0

Monilia sp. MMTL 2887 0 0 0 0 0 0 0 0

Nigrospora oryzae MMTL 2897 0 8.66±0.57 0 0 0 0 0 0

Penicillium sp. 1 MMTL 2967 15.33±0.33 0 0 0 12.33±0.66 0 0 0

Penicillium sp. 2 MMTL 3054 13.00±0.57 8.33±0.33 0 0 0 0 0 0

T. viride MMTL 3112 0 0 0 0 0 0 0 0

Veronaea musae MMTL 3120 0 0 0 0 0 0 0 0

A. tubingensis (JQ031155) MMTL 3130 0 0 0 0 0 0 0 0

C. crassipes (JQ031156) MMTL 3150 0 0 0 0 0 0 0 0

B. rhodina (JQ031157) MMTL 3177 31.33±0.33 33.00±0.88 40.33±0.33 32.66±0.66 34.33±0.33 45.66±0.33 12.83±0.16 36.66±0.33

A. sydowii (JQ031158) MMTL 3215 0 0 0 0 0 0 0 0

P. violaceum (JQ031159) MMTL 3251 13.30±0.66 0 0 0 0 0 0 0

Controls

Methanol 0 0 0 0 0 0 0 0

Ampicillin (10 μg/disk) 0 0 0 0 0 0 0 0

Ciprofloxacin (5 μg/disk) 18.00±0.00 39.00±0.00 40 40 34.00±0.00 32.00±0.00 30 32.00±0.00

aA, Shigella flexnii IMS/GN1; B, E. coli ATCC 25922; C, Salmonella enteritidis IMS/GN3; D, S. paratyphi IMS/GN4; E, Pseudomonasaeruginosa ATCC 27853; F, Citrobacter freundii IMS/GN5; G, Morganella morganii IMS/GN6; H, Proteus vulgaris IMS/GN7

Endomycoflora of Tinospora cordifolia 395

work done by Schulz et al. [54]. We found B. rhodina (JQ031157 “MMTL-3177”) and C. globosum to be activeagainst all pathogens tested, with B. rhodina exhibiting thehighest antibacterial activity comparable to standard drugciprofloxacin. F. oxysporum, C. dematium, and Penicilliumsp. 1 and 2 also showed significant activity against patho-gens (Table 3).

Among the most frequently isolated taxa in this study, C.globosum, Colletotrichum, and Penicillium species have thegreatest potential to produce interesting secondary metabo-lites. Bioactive compounds such as chaetoglobosins, chaeto-pyranin, and globosumones A–G, have been isolatedsuccessfully from an endophytic Chaetomium sp. and metab-olites from various Chaetomium isolates showed cytotoxicactivities against human cancer cell lines [36]. ChaetoglobosinB showed weak antibacterial activity against Staphylococcusaureus and Escherichia coli [42]. Metabolites of endophyticChaetomium spp. inhibited plant pathogens, e.g., Pyreno-phora tritici-repentis on wheat [32] or powdery mildew onbarley [66]. Similarly, various species of Colletotrichum arewell-known sources of novel bioactive secondary metabolites[17, 74]. An endophytic Colletotrichum sp. from Artemisiaannua was shown to release an oligosaccharide elicitor medi-ating biosynthesis and accumulation of the antimalarial drugartemisinin [68] and secondary metabolites of a Colletotri-chum sp. from Ilex canariensis showed antimicrobial activityagainst Microbotryum violaceum (fungi), Chlorella fusca (al-gae), E. coli and Bacillus megaterium (bacteria) [73]. Endo-phytic Penicilllium spp. are well known to produce a plethoraof secondary metabolites with antibiotic (bactericidal, fungi-cidal, or insecticidal) and/or cytotoxic, anticancer activity [29,52, 69]. Some endophytic Penicillium species also produceplant growth hormones such as gibberellins [25, 35].

The endophytes isolated during this study offer an excel-lent platform for the discovery of novel bioactive (antibac-terial and antifungal) compounds. Presently, we are engagedin isolation and characterization of the active principle ofcrude extract from potential endophytic fungi received dur-ing this study.

Acknowledgment Authors are highly thankful to Head and Coordi-nator (Prof. BR Chaudhary), CAS in Botany, BHU, Varanasi, forproviding the facilities to carry out the research. The authors thank toCSIR and UGC for Financial Support in the form of JRF and SRFs.Financial assistance to RNK from DST, New Delhi, is gratefullyacknowledged (file no. SR/SO/PS-78-2009, dt-10-5-2010).

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