Endophytic Mycobiota Characterization of the Amazonian Mistletoe <i>Cladocolea micrantha</i> Hosted in Cashew Tree

doi:10.4236/ajps.2013.44113

Paper Menu >>

Journal Menu >>

American Journal of Plant Sciences, 2013, 4, 917-921

http://dx.doi.org/10.4236/ajps.2013.44113 Published Online April 2013 (http://www.scirp.org/journal/ajps)

917

Endophytic Mycobiota Characterization of the Amazonian

Mistletoe Cladocolea micrantha Hos ted in Cashew Tree

Anderson C. Guimarães1, Antonio C. Siani2*, José L. Bezerra3, Antonia Q. Lima de Souza4,

Maria Inez M. Sarquis5

1Instituto de Ciências Exatas e Tecnologia de Itacoatiara, Universidade Federal do Amazonas, Itacoatiara, Brazil; 2Departamento de

Produtos Naturais, Farmanguinhos, Fiocruz, Rio de Janeiro, Brazil; 3Departamento de Ciências Agronômicas e Ambientais,

Universidade Estadual de Santa Cruz, Ilhéus, Brazil; 4Laboratório de Genética, Escola Superior de Ciências da Saúde, Universidade

Estadual do Amazonas, Manaus, Brazil; 5Laboratório de Taxonomia Bioquímica e Bioprospecção de Fungos, Instituto Oswaldo Cruz,

Fiocruz, Rio de Janeiro, Brazil.

Email: *[email protected]

Received March 5th, 2013; revised April 3rd, 2013; accepted April 12th, 2013

License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

ABSTRACT

Endophytic fungi were identified from different parts of the medicinal parasitic mistletoe Cladocolea micrantha and

from its host Anacardium occidentale, suggesting a strict host-parasite relationship. Eight fungal endophytes were iso-

lated and morphologically characterized. The ascomycete Guignardia mangiferae and strains of Mycelia sterilia were

prevalent in the isolations. The unequivocal identification of Guignardia mangiferae at a probabilistic degree close to

100% was carried out by DNA extraction followed by PCR analyses of the ITS-1 and ITS-2 regions and comparison of

the genetic sequence with the NCBI database.

Keywords: Cladocolea micrantha; Endophytic mycobiota; Loranthaceae; Guignardia mangiferae; Anacardium

occidentale

1. Introduction

The Loranthaceae mistletoes, popularly known in Brazil

as “erva-de-passarinho” [1] play an important and com-

plex part in the biological system they inhabit, interacting

with insects, birds, fishes and mammals [2]. These epi-

phytic and hemiparasitic plants adhere to the branches

and twigs of trees by means of haustoria, penetrating the

host to absorb water and nutrients [3]. Species of this

family are used all over the world in traditional medicine

to treat a wide range of diseases, such as arthritis, diabe-

tes, general inflammation, breathing and nervous prob-

lems and some types of cancer [4]. In the Amazonian

region, the leaves of the species Cladocolea micrantha

(Eichler) Kuijt are widely used in the non-conventional

therapy of several types of tumors and inflammatory

processes [5], with a preference for specimens growing

on the cashew tree Anacardium occidentale (Anacardi-

aceae) [6]. The genus Cladocolea comprises approxi-

mately 40 species and occurs from central to southern

Mexico and in some localities of Latin America. The

most important morphological feature of this genus is the

occurrence of a determinate inflorescence and single lat-

eral ebracteolate flowers or a simple derivative thereof;

this characteristic serves to differentiate Cladocolea from

other similar mistletoe genera [7]. Chemical studies on

this genus are restricted to Cladocolea micrantha which

describes the isolation of diverse triterpenes as well as

kaempferol and quercetin derivatives presenting an un-

common interglycosidic O-(1→3) linkage [8]. Comple-

mentarily, the triterpenes α-amyrin, β-amyrin and their

respective ketones were remarkably characterized from

the chloroform extracts of mycelia of G. mangiferae, P.

clavisporus and P. guepinii 7-d to 14-d-old colonies, iso-

lated from this mistletoe [9].

Endophytic fungal communities occupy intercellular

organs in plants, usually inducing an associative rela-

tionship that ranges from mutualism to host plant para-

sitism. Such a symbiosis may confer some protection

against insects, herbivores or phytopathogenic microor-

ganisms by producing toxins, antibiotics, specific en-

zymes or growth-promoting agents and other substances

[10]. Endophytic fungi have also been found to be even-

*Corresponding author.

Endophytic Mycobiota Characterization of the Amazonian Mistletoe Cladocolea micrantha Hosted in Cashew Tree

918

tually responsible for the bioactive compounds produced

by host plants. A corollary to this assumption would be

the production of the anti-cancer agent taxol by the

endophytic fungi Taxomyces andreanae, Pestalotiopsis

microspora and P. guepinii isolated from the Pacific

Taxus brevifolia. Similarly, the fungus Phialocephala

fortinii isolated from Podophyllum peltatum may pro-

duce the antitumoral podophyllotoxin [11,12]. Endo-

phytes have been identified on species associated with

many botanical families, and currently, a significant

amount of research is focused on obtaining active me-

tabolites from endophytes, aiming to utilize them in

medicine and biological control [11,13,14]. The present

study aimed to identify and to morphologically and mo-

lecularly characterize the endophytic fungi from different

parts of the parasite mistletoe Cladocolea micrantha and

from its host Anacardium occidentale.

2. Material and Method

2.1. Plant Material

Leaves and stems of Cladocolea micrantha (Eichler)

Kuijt and Anacardium occidentale L. for the host-para-

site mycobiotic analyses were collected on the campus of

the Federal University of Amazonas (UFAM), Manaus,

Brazil, S 03˚07.785', W 058˚26.631', each from a single

individual host specimen 8 m tall. Both species were

identified, and vouchers were deposited in the Herbarium

of UFAM under No. 6213.

2.2. Surface-Disinfecting and Inoculation of the

Botanic Material

Endophytic fungi were isolated according to the proce-

dure by Petrini (1992) [15,16] using fresh botanical ma-

terial from both C. micrantha and A. occidentale. The

plant material was carefully washed with a neutral deter-

gent and running water. After drying, the leaves and

stems were sliced into pieces of approximately 8 cm2 and

were soaked for 1 min in a 500 ml beaker containing

70% ethanol in water. The fragments were then trans-

ferred to another beaker containing a 3% solution of so-

dium hypochlorite for a period of 4 min. The pieces of

leaves and stems were then returned to the beaker con-

taining the ethanol/water solution for 30 s and were fi-

nally transferred to a flask with sterilized water for 1 min.

After this procedure, fragments of leaves and stems from

both plants were cultured on eight Petri plates (six frag-

ments per plate) containing malt-agar extract (MAE)

(Difco) and chloramphenicol (100 μg/ml MAE) with the

aid of a Drigalski loop, resulting in 192 inocula distrib-

uted onto 32 plates. A negative control was run by ap-

plying 0.05 ml of the distilled water from the last sterili-

zation step onto Petri dishes using the same medium.

Cultured samples were incubated at room temperature

(approximately 25˚C in the laboratory) and observed

daily.

2.3. Endophytic Fungi Isolation and

Identification

After eleven days, the plates contained 160 growing

colonies that were singly prepared on slides and carefully

inspected under a microscope. Identical fungal strains

were separately pooled into eight different representative

groups according to their macro- and micromorphologi-

cal similarities, and their purity was certified by macro-

and microscopic evaluation. For genus and species as-

signments, the fungal strains were separately grown on

the following media: PDA, malt agar extract (Difco),

Czapek-dox agar (Merck) and oatmeal agar (60.0 g oat-

meal and 18.0 g agar). Nutrient-poor synthetic medium

agar [1.0 g KH2PO4, 1.0 g KNO3, 0.5 g MgSO4·7H2O,

0.5 g KCl, 0.2 g glucose, 0.2 g sucrose and 18.0 g agar

(all Merck) in 1000 ml distilled water] and sunflower

seed extract agar were also used according to classical

methodologies [17]. The microscopic characteristics were

evaluated in microcultures on coverslips in a drop of

Amann’s lactophenol [20.0 g phenol (Quimibras), 20.0

ml lactic acid (Merck), 20.0 ml distilled water, 40.0 ml

glycerine (Merck)] with 0.05 g cotton blue (Sigma). The

filamentous fungi were classified based on standard pro-

cedures reported for morphological characterization [18-

20]. The fungi were then transferred to test tubes con-

taining MAE for successive growth. Samples were ly-

ophilized and stored under mineral oil in the Fungi Cul-

ture Collection Filamentous Fungi of the Oswaldo Cruz

Institute, Rio de Janeiro, under Nos. IOC4553 (Aspergil-

lus sp.), IOC 4548 (G. mangiferae), IOC4555 (M. ster-

ilia), IOC4549 (Myrothecium sp.), IOC4550 (P. clavis-

porus), IOC4551 (P. guepinii), IOC4552 (T. pseudoviri-

dae) and IOC4554 (Drechslera sp.).

2.4. DNA Extraction and PCR Conditions

G. mangiferae identification was confirmed by sequenc-

ing the ITS-1 and ITS-2 rDNA and comparing it with

sequences from GenBank. For ITS-1 and ITS-2 rDNA

analyses, the strain was stirred at room temperature for

12 days (120 rpm) on a potato-dextrose medium enriched

with 0.2% PDY (potato dextrose yeast) extract. From

200 mg of mycelium separated by filtration, genomic

DNA was extracted using the cetyltrimethylammonium

bromide method [21]. Prior to PCR, the DNA concentra-

tion was visually estimated at 100 ng/μl by comparing it

with the 1 kb marker on a 1% agarose gel. Each 25 μl of

reaction contained 10.2 μl of water, 2.5 μl of 10× buffer,

3.0 μl of 25 mM MgCl2, 3.0 μl of 1.25 mM dNTP, 2.0 μL

each of ITS-1 and ITS-2 primers at 10 mM, 0.3 μl of Taq

Endophytic Mycobiota Characterization of the Amazonian Mistletoe Cladocolea micrantha Hosted in Cashew Tree

919

DNA polymerase (5.0 U) and 2.0 μl of genomic DNA.

Four reactions were carried out with an initial denaturing

step of 94˚C for 4 min, followed by 40 cycles of 2 min

denaturing at 94˚C, 2 min annealing at 55˚C and 2 min

elongation at 72˚C. The amplification was terminated for

10 min at 72˚C. PCR products were analyzed by electro-

phoresis on 1.5% agarose gels stained with ethidium

bromide, compared with a DNA ladder of 100 bp and

estimated at 50 ng/μl. In all, 2 μl of the PCR products

were sequenced on an acrylamide gel, and the sequences

obtained were matched with those deposited in the NCBI

(National Center for Biotechnology Information) data-

base.

3. Results and Discussion

A total of 160 fungal strains (from the 192 total frag-

ments of plant material) comprising 8 groups of endo-

phytic fungi were isolated from both mistletoe and the

plant host. Stems and leaves of the mistletoe Cladocolea

micrantha led to the isolation of the following fungal

species and genera: Aspergillus sp. (Trichocomaceae,

Order Eurotiales), Guignardia mangiferae (Botryosphae-

riaceae, Order Botryosphaeriales) Mycelia sterilia (deu-

teromycetes, Order Agonomycetales), Myrothecium sp.

(Incertae sedis, Order Hypocreales), Paecilomyces clav-

isporus (Trichocomaceae, Order Eurotiales), Pestalo-

tiopsis guepinii (Amphisphaeriaceae, Order Xylariales)

and Trichoderma pseudoviridae (Hypocreaceae, Order

Hypocreales). The following species and genera were

identified in the tissues of Anacardium occidentale:

Pestalotiopsis guepinii, Drechslera sp. (Pleosporaceae,

Order Pleosporales), Guignardia mangiferae, Mycelia

sterilia, and Trichoderma pseudoviridae. The three spe-

cies/genera in boldface were common to both the parasite

and the host plant (Ta ble 1). All of these occurrences are

described here for the first time, especially for the

Cladocolea micrantha mycobiota, for which, as far as we

know, this is the first report.

The non-sterile ascomycetes G. mangiferae and Myce-

lia sterilia were found as the most frequent fungal endo-

phytes in both the parasite and the host plants in the ex-

perimental sampling. Identification of G. mangiferae was

based on macroscopic characteristics and on the micro-

scopic measurements of individuals from the 7-d-old

colony on MAE, which presented the following dimen-

sions: pseudothecia [(640 - 1232) × (112 - 424) μm];

ostioles [(80 - 400) × (80 - 200) μm]; asci [(40 - 68) ×

(10 - 12) μm; ascospores [(15 - 16) × (5 - 6) μm] [22].

Similar results for the ascospore sizes [(13 - 17) × (5 - 8)

μm] were observed for G. mangiferae isolated from Ana-

cardium giganteum [23]. The trusted pairwise compari-

sons revealed 99% identity with G. mangiferae and 100%

reliability for the 576 DNA base pairs (data not shown).

Such a high probabilistic correlation provides additional

relevant data on tropical endophytes, given that the Gui-

gnardia species present wide intraspecific genetic vari-

ability [24,25]. Species of Guigna rdia are usually recog-

nized as endophytic fungi and have been isolated from

asymptomatic tissues from a diversity of Myrtaceae and

Anacardiaceae species as well as citrus plants [25]. How-

ever, some species such as G. citricarpa and G. psidii

cause serious diseases to agriculture by infesting fruit

and leaves post-harvest. General studies of endophytes

are more prevalent in temperate regions [26] and should

be extended to tropical species when considering the

pharmaceutical potential of tropical medicinal plants [27].

4. Conclusion

The findings reported here reinforce the cosmopolitan

occurrence of the endophytic G. mangiferae in tropical

plants [23,28,29]. The non-sterile mycobiota of the leaves

of both parasitic and host plants is characterized by the

common presence of G. mangiferae as the most frequently

occurring fungus. This presence was not observed for the

Table 1. Endophytic fungi characterized in the tissues of Cladocolea micrantha and Anacardium occidentale.

Fungus species CM leaf CM stem AO leaf AO stem

Aspergillus sp. [2] n n 2/10 n

Drechslera sp. [1] n n 1/10 n

Guignardia mangiferae [15] 8/15 n 7/10 n

Myrothecium sp. [2] 1/15 1/18 n n

Mycelia sterilia [133] 31/15 38/18 35/10 29/3

Paecilomycesclavisporus [2] 2/15 n n n

Pestalotiopsis guepinii [2] n 2/18 n n

Trichoderma pseudoviridae [3] 1/15 n 1/10 1/3

CM = Cladocolea micrantha; AO = Anacardium occidentale. In brackets = total of similar pooled strains. Results (X/Y) indicate the ra-

tio of culture plates (X) growing positively over the total cultured plates (Y), including replication experiments; n = no growth observed.

Endophytic Mycobiota Characterization of the Amazonian Mistletoe Cladocolea micrantha Hosted in Cashew Tree

920

stems, suggesting that some horizontal fungal contami-

nation (leaf-leaf) cannot be excluded [30]. This is the

first description of G. mangiferae as an endophyte in C.

micrantha. Moreover, its presence on A. occidentale sug-

gests a close host-parasite relationship, a fact that may be

relevant to the medicinal properties of the parasite plant

and to the popular preference for G. mangiferae grown

on this host plant.

5. Acknowledgements

The authors wish to thank Dra. Lilian A. Procópio for the

plant identification, and plant collectors Silo Silva and

Rosalba Bilby for their help.

REFERENCES

[1] H. Lorenzi, “Plantas Daninhas do Brasil: Terrestres, Aquá-

ticas, Parasitas e Tóxicas,” 3rd Edition, Instituto Plant-

arum, Nova Odessa, 2000.

[2] D. M. Watson, “Mistletoe—A Keystone Resource in

Forest and Woodlands Worldwide,” Annual Review of

Ecological System, Vol. 32, 2001, pp. 219-249.

doi:10.1146/annurev.ecolsys.32.081501.114024

[3] J. E. L. S. Ribeiro, M. J. G. Hopkins, A. Vicentini, C. A.

Sother, M. A. S. Costa, J. M. Brito, M. A. D. Souza, L. H.

P. Martins, L. G. Lohmann, P. A. C. L. Assunção, E. C.

Pereira, C. F. Silva, M. R. Mesquita and L. C. Procópio,

“Flora da Reserva Ducke: Guia de Identificação das

Plantas Vasculares de uma Floresta de Terra-Firme na

Amazônia Central,” Instituto Nacional de Pesquisas da

Amazônia, Brazil, 1999.

[4] A. C. Guimarães, “Estudo Químico e Biológico de Cla-

docolea micrantha (Loranthaceae), uma planta medicinal

da região Amazônica,” Doctorate Thesis, Universidade

Federal do Rio de Janeiro, Brazil, 2005.

[5] A. Cáceres, “Plantas de Uso Medicinal en Guatemala,”

Editorial Universitaria, San Carlos de Guatemala, 1996.

[6] J. Kuijt, “Inflorescence Structure and Generic Placement

of Some Small-Flowered Species of Phthirusa (Loran-

thaceae),” Systematic Botany, Vol. 16, No. 2, 1991, pp.

283-291. doi:10.2307/2419280

[7] A. C. Guimarães, R. M. Kuster, A. C. F. Amaral, J. L. P.

Ferreira and A. C. Siani, “Histological Study of the Leaf

and Stem of the Amazonian Medicinal Mistletoe Cla-

docolea micrantha (Loranthaceae),” International Jour-

nal of Botany, Vol. 3, No. 2, 2007, pp. 218-221.

doi:10.3923/ijb.2007.218.221

[8] A. C. Guimarães, A. Magalhães, M. J. Nakamura, A. C.

Siani, A. L. F. Sampaio and C. Barja-Fidalgo, “Flavon-

oids Bearing an O-arabinofuranosyl-(1→3)-Rhamnoside

Moiety from Cladocolea micrantha: Inhibitory Effect on

Human Melanoma Cells,” Natural Product Communica-

tions, Vol. 7, No. 10, 2012, pp. 1311-1314.

[9] A. C. Guimarães, R. M. Kuster, I. Neves Jr., M. C. S.

Lourenço and M. I. Sarquis, “Preliminary Investigation

on the Mycelial Composition and Antimicrobial Potential

of Endophytic Fungi from the Amazonian Mistletoe Cla-

docolea micrantha (Eichler) Kuijt (Loranthaceae),” Re-

vista Fitos, Vol. 4, No. 2, 2009, pp. 90-101.

[10] A. V. Sturtz, B. R. Christie, B. G. Matheson, W. J.

Arsenault and N. A. Buchanan, “Endophytic Bacterial

Communities in the Periderm of Potato Tubers and Their

Potential to Improve Resistance to Soil-Borne Plant

Pathogens,” Plant Patholology, Vol. 48, No. 3, 1999, pp.

360-369. doi:10.1046/j.1365-3059.1999.00351.x

[11] G. Strobel and B. Daisy, “Bioprospecting for Microbial

Endophytes and Their Natural Products,” Microbiology

and Molecular Biological Reviews, Vol. 67, No. 4, 2003,

pp. 491-502. doi:10.1128/MMBR.67.4.491-502.2003

[12] A. L. Eyberger, R. Dondapati and J. R. Porter, “Endo-

phyte Fungal Isolates from Podophyllum peltatum Pro-

duce Podophyllotoxin,” Journal of Natural Products, Vol.

69, No. 8, 2006, pp. 1121-1124. doi:10.1021/np060174f

[13] G. Strobel, B. Daisy, U. Castillo and J. Harper, “Natural

Products from Endophytic Microorganisms,” Journal of

Natural Products, Vol. 67, No. 2, 2004, pp. 257-268.

doi:10.1021/np030397v

[14] K.-H. Kogel, P. Franken and R. Hückelhoven, “Endo-

phyte or Parasite—What Decides?” Current Opinion in

Plant Biology, Vol. 9, No. 4, 2006, pp. 358-363.

doi:10.1016/j.pbi.2006.05.001

[15] O. Petrini, T. N. Sieber, L. Toti and O. Viret, “Ecology,

Metabolite Production and Substrate Utilization in Endo-

phytic Fungi,” Natural Toxins, Vol. 1, No. 3, 1992, pp.

185-196. doi:10.1002/nt.2620010306

[16] A. Q. L. Souza, A. D. L. Souza, S. Astolfi-Filho, M. L.

B. Pinheiro, M. I. M. Sarquis and J. O. Pereira, “Ati-

vidade Antimicrobiana de Fungos Endofíticos Isolados de

Plantas Tóxicas da Amazônia: Palicourea longiflora

(Aubl.) Rich. e Strychnos cogens Bentham,” Acta Ama-

zonica, Vol. 34, No. 2, 2004, pp. 185-195.

doi:10.1590/S0044-59672004000200006

[17] S. T. Jaronski and R. C. Axtell, “Simplified Production

System for the Fungus Lagenidium giganteum for Opera-

tional Mosquito Control,” Mosquito News, Vol. 44, No. 3,

1984, pp. 377-381.

[18] M. B. Ellis, “More Dematiaceous Hyphomycetes,” CAB

Press, Kew, 1976.

[19] B. C. Sutton, “The Coelomycetes: Fungi Imperfecti with

Pycnidia, Acervuli and Stromata,” Commonwealth My-

cological Institute, Kew, 1980.

[20] J. J. Pitt, “A Laboratory Guide to Common Penicillium

Species,” Academic Press, Autralia, 1985.

[21] T. J. White, T. L. Bruns, S. Lee and J. W. Taylor, “Am-

plication and Direct Sequencing of Fungal Ribosomal

RNA Genes for Phylogenetics,” In: M. Innis, D. H. Gel-

fand, J. J. Sninsky and J. T. White, Eds., PCR Protocols:

A Guide to Methods and Applications, Academic Press,

Waltham, 1990, pp. 315-322.

[22] A. J. Roy, “Some Fungi from Almora,” Indian Phytopa-

thology, Vol. 20, No. 21, 1968, pp. 340-348.

[23] K. F. Rodrigues, T. N. Sieber, C. R. Grünig and O.

Holdenrieder, “Characterization of Guignardia mangif-

erae Isolated from Tropical Plants Based on Morphology,

ISSR-PCR Amplifications and ITS1-5.8S-ITS2 Sequen-

Endophytic Mycobiota Characterization of the Amazonian Mistletoe Cladocolea micrantha Hosted in Cashew Tree 921

ces,” Mycological Research, Vol. 108, No. 1, 2004, pp.

45-52. doi:10.1017/S0953756203008840

[24] C. Glienke-Blanco, C. I. Aguilar-Vildoso, M. L. C. Vieira,

P. A. V. Barroso and J. L. Azevedo, “Genetic Variability

in the Endophytic Fungus Guignardia citricarpa Isolated

from Citrus Plants,” Genetic and Molecular Biology, Vol.

25, No. 2, 2002, pp. 251-255.

doi:10.1590/S1415-47572002000200021

[25] E. Wickert, A. Góes, E. G. M. Lemos, A. Souza, E. L.

Silveira, F. D. Pereira and D. Rinaldo, “Relações Filoge-

néticas e Diversidade de Isolados de Guignardia spp.

Oriundos de Diferentes Hospedeiros nas Regiões ITS1-5,

8s-ITS2,” Revista Brasileira de Fruticultura, Vol. 31, No.

2, 2009, pp. 360-380.

doi:10.1590/S0100-29452009000200010

[26] N. S. Raviraja, “Fungal Endophytes in Five Medicinal

Plant Species from Kudremukh Range, Western Ghats of

India,” Journal of Basic Microbiology, Vol. 45, No. 3,

2005, pp. 230-235. doi:10.1002/jobm.200410514

[27] G. Strobel, B. Daisy and U. Castillo, “The Biological

Promise of Microbial Endophytes and Their Natural

Products,” Plant Patholology Journal, Vol. 4, No. 2,

2005, pp. 161-176. doi:10.3923/ppj.2005.161.176

[28] R. P. Baayen, P. J. Bonants, G. Verkley, G. C. Carroll, H.

A. van der Aa, M. de Weerdt, I. R. van Brouwershaven, G.

C. Schutte, W. Maccheroni, C. G. de Blanco and J. L.

Azevedo, “Nonpathogenic Isolates of the Citrus Black

Spot Fungus, Guignardia citricarpa, Identified as a Cos-

mopolitan Endophyte of Woody Plants, Guignardia Man-

giferae (Phyllosticta capitalensis),” Phytopathology, Vol.

92, No. 5, 2002, pp. 464-477.

doi:10.1094/PHYTO.2002.92.5.464

[29] M. V. Tejesvi, M. S. Nalini, B. Mahesh, H. S. Prakash, K.

R. Kini, H. S. Shetty and V. Subbiah, “New Hopes from

Endophytic Fungal Secondary Metabolites,” Boletin de la

Sociedad Química de México, Vol. 1, No. 1, 2007, pp. 19-

26.

[30] A. M. M. N. Pirttil, “Endophytes in the Buds of Scots

Pine (Pinus silvestres L.),” M.Sc. Dissertation, University

of Oulu, Oulu, 2001.