A study was undertaken to identify the presence of fungal endophyte in fern plant, Psilotum nudum. Being the only living

species of a once populated division, P. nudum is the most primitive vascular plant. Cross section of stipe and rhizoid parts

was done to observe the presence of endophyte within parenchyma cells. Samples of P. nudum were collected at different

sites in Bangi. Anatomical study showed P. nudum has percentage of infected cells highest in rhizoid parenchyma cortex

(22.2%) and lowest in the apical area (3.1%). Eleven fungal endophyte isolates were successfully isolated and identified

from P. nudum with Aspergillus being the major genus. Is Aspergillus a host-specific endophyte? The present study shows

the compatibility of P. nudum parenchyma cells as a host to fungal endophyte

Affina E.,

Nazlina Ibrahim,

Noraini Talip,

UKM Journal Article Repository

* To whom correspondence should be addressed.ANATOMY OF SYMBIOTIC FUNGAL ENDOPHYTES INPsilotum nudum (L.) P. BeauvAFFINA, E.1, NORAINI, T.1* and NAZLINA, I.21School of Environmental and Natural Resource Sciences,2School of Biosciences and Biotechnology, Faculty of Science and Technology,Universiti Kebangsaan Malaysia, 43600 Bangi, Selangor.*Email: norainitalip@gmail.com/ntalip@ukm.edu.myABSTRACTA study was undertaken to identify the presence of fungal endophyte in fern plant, Psilotum nudum. Being the only livingspecies of a once populated division, P. nudum is the most primitive vascular plant. Cross section of stipe and rhizoid partswas done to observe the presence of endophyte within parenchyma cells. Samples of P. nudum were collected at differentsites in Bangi. Anatomical study showed P. nudum has percentage of infected cells highest in rhizoid parenchyma cortex(22.2%) and lowest in the apical area (3.1%). Eleven fungal endophyte isolates were successfully isolated and identifiedfrom P. nudum with Aspergillus being the major genus. Is Aspergillus a host-specific endophyte? The present study showsthe compatibility of P. nudum parenchyma cells as a host to fungal endophyte.Key words: Endophyte; anatomy; Psilotum nudum; parenchymaINTRODUCTIONPlants interact with endophytes in mutualism andparasitism association, forming a complexrelationship in time and space (Herrera et al., 2002).Traditionally, ecological study of the relationshipis examined separately by considering the pairinginteraction between plants and pollinators such as,plants with mycorrhizal fungi and plants withparasites. However, this approach has changedcompletely in the last few years with the increasingnumber of studies that emphasize the importance ofmultiple interactions, including biota relations onthe ground and give an understanding of theecological and evolutionary processes in nature(Gehring & Bennett, 2009). However, therelationship that exists between plant parasites,mycorrhizal hosts are still less studied.A plant relationship with mycorrhizal fungi isone of the most important relationships and isknown as symbiosis. The type of interactioninvolved is mutualism which refers to the benefitgained by both parties (Elizabeth 1972). Plants areused as their host to live, they develop strategies inthe chemical aspects that favour their existence. Inexchange for carbohydrates from the plants, theendophytic fungi alter plant nutrient content andenhance the production of secondary metabolites.Changes in chemical composition act to prevent theplant from being attack by insects or pests. (Jeffreyet al., 2008). Endophytes usually inhabit aboveground tissues like leaves, stem and bark whichdifferentiates them from mycorrhizal symbionts.However, the differentiation is not strong becauseendophytes also can live in root tissue. Overall,endophytic fungi are ubiquitous and extremelydiverse in host plants (Arnold et al., 2003).Psilotum nudum is an ornamental fern and alsopopular in traditional medicine (Figure 1 A & B).The benefits of P. nudum vary according to theirparts. Oily spores of the species are given to infantsto stop diarrhoea. The herb’s juice showedantibacterial activity against Micrococcus pyogenesand Pseudomonas aeruginosa and also used as apurgative (Kumari et al., 2011). Yamazaki et al.(2001) stated P. nudum contains flavonoids whichhave been associated with medicinal value onreduction of chronic disease (Ramos 2007).Flavonoids are a large family of plant secondarymetabolites, principally recognized for their health-promoting properties in human diets (Hernendez etal., 2009). The objectives of this study are toidentify the parts of P. nudum consisting the highestpercentage of fungal endophytes, to observe themodification of anatomical characteristic due to theMalays. Appl. Biol. (2015) 44(4): 11–1812 ANATOMY OF SYMBIOTIC FUNGAL ENDOPHYTES IN Psilotum nudum (L.) P. Beauvpresence of fungal endophyte and to identify thefungal endophyte present.MATERIALS AND METHODSPercentage of endophyte presentFresh samples of the whole plant of P. nudumwere taken from three different localities, TamanPaku Pakis UKM, (originally from Banting,Selangor), Rumah Tumbuhan UKM and BandarBaru Bangi. A list of species studied is given inTable 1. Voucher specimens were kept at UKMBHerbarium. In this study, P. nudum were divided intofive sections namely apex aerial (aa), below apexaerial (baa), middle aerial (ma), bottom aerial (ba)and rhizoid (rh) (Figure 1C). Specimens were fixedin acetic acid solution (1:3, acetic acid: 70%alcohol). Fixation, embedding and sectioning wereperformed following the methods described byJohansen (1940) and Sass (1958) while sectioningwas done using a sliding microtome. Sections werestained in Safranin and Alcian Blue and thendehydrated in an alcohol series of 50%, 70%, 95%,and 100%. Slides were mounted in Euparal, thensections of the specimen were photographed usingan Olympus Diaphlan microscope and images wereprocessed using Analysis Docu Software (Johansen1940; Saas 1958). Anatomical descriptions followedOgura (1972) and the percentage of endophytic cellsis counted by the modification formula of stomatacounting by Metcalfe and Chalk (1957). The formulais based on the appearance of hypha endophytewhich explained using this formula:Percentage of endophytic cells:Cells with endophyte  x 100Cells with endophyte + Cells without endophytesIsolation of fungiFungal isolation was  done by using a surfacesterilization method as proposed by Petrini et al.(1992) and sliced section (10mm x 10mm) was laidon Potato Dextrose Agar (PDA). Incubation wasdone for five days at 30°C. Lactophenol cotton bluewet mount preparation is used to demonstratefungi and fungal elements under microscope.Characterisation of endophytic fungi was carriedout by macroscopic and microscopic observations.Parameters for macroscopic observation includecolony color and diameter, pigmentation, presenceof pigment diffusion in agar, mycelium texture andcolony edge. Microscopic observation at 400xmagnification was done to observe the structure andpresence of hyphae, septa end, asexual spores andsexual spores.RESULTS AND DISCUSSIONPercentage cells with fungal endophyte presenceThe sections were calculated according to thefrequent appearance of hyphae (h) and hyphal coil(hc) in parenchyma tissue. Based on result, it showsthat frequency of endophytes presence is highest inthe rh part. High density fungal endophytes colonieswere observed in the baa, ma, ba and rh parts. Rhpart showed to have the highest percentage ofendophytes colonization by 22.22% compared toothers (Table 2). The abundance of endophytes atthe rh correlates with the position of this structurewhich is closest to the ground. This highcolonization of endophytic fungi can be consideredof the spread and dominance of fungal endophytesthrough soil nutrient uptake (Rodriguez et al.,2008). Therefore, it is possible that a sign ofpossibility that fungal endophytes found in P.nudum are strongly derived from the surroundinghabitat or area. The presence of fungal endophyteshas been previously reported by Peterson et al.(1981) on the gametophyte of P. nudum that foundabundant fungal endophyte in rhizoid and corticolparenchyma in most cells. Findings from this studyagreed with Vega et al. (2010) where the highestfrequency of colonization by endophytes is at therhizoid.Results have shown that parenchyma cellscontaining endophytes normally have thicker cellwalls. This is due to the structure of the haustoriumhyphae that penetrate through the pores of the wallin parenchyma cells, in order to absorb water andnutrients from the host (Bhandari & Mukherji 1993).Admission hyphae and mycelium structureaccordingly will improve parenchyma cells wallTable 1. List of species studiedNo. Species Locality Part used Date of collection & collector  1 P. nudum AF1 Taman Paku Pakis UKM Aerial & rhizoid 05.05.11 Ruzi Rahman Affina Eliya(Bangi, Selangor)  2 P. nudum AF2 Rumah Tumbuhan, UKM Aerial & rhizoid 05.05.11 Ruzi Rahman Affina Eliya  3 P. nudum AF3 Bandar Baru Bangi area Aerial & rhizoid 05.05.11 Ruzi Rahman Affina EliyaANATOMY OF SYMBIOTIC FUNGAL ENDOPHYTES IN Psilotum nudum (L.) P. Beauv 13Table 2. Percentage density of endophyte in different part of Psilotum nudumNo. Parts Cell with endophyte Cell without endophyte Percentage (%)  1 Apex aerial (aa) 10 311 3.1  2 Below apex aerial (baa) 22 549 3.8  3 Middle aerial (ma) 54 623 7.9  4 Bottom aerial (ba) 60 412 12.7  5 Rhizoid (rh) 94 329 22.2Fig. 1. Psilotum nudum (L.) P. Beauv. (A) Aerial Morphology, (B) Synangium.Source: Taman Paku Pakis UKM Bangi) (C) Diagram of plant parts.14 ANATOMY OF SYMBIOTIC FUNGAL ENDOPHYTES IN Psilotum nudum (L.) P. Beauvthickness. It has also been reported by Vega et al.(2007) which stated that endophytic parenchymacells were observed in the thickened parenchymacells wall. Observations on thin-walled parenchymacells also did not show the presence of anystructures and no endophytic hyphae were observedto dominate the area.Parenchyma cells that contain endophytes wereobserved to have dark cells. These factors caused byfungal endophytes cells that absorb more AlcianBlue staining solution compared to those cellswithout endophytes. Based on the study of Vega etal. (2007), the cells cross section of Cytinussalviifolius (angiosperm) in the presence ofendophytes, visibly coloured dark blue more thanother cells in C. salviifolius that not containendophytic hyphae structures. Observations todetermine the presence of endophytes inparenchyma cells can simply be made by comparingthe cell color, cell sizes and also confirmation ofattendance through observation under a lightmicroscope with high magnification.Anatomical features are characterised by cells,tissues or internal structure of specific plants species.In the species studied, anatomical features are thepresence of wide parenchyma cells with intercellularspaces. These anatomical characteristics areconsidered to have some sort of influence andimpact on the presence of fungal endophytes(Figure 2). It has also been discovered by more than100 years of research that the habitat where the plantspecies grow and breed also influenced the presenceof endophytic fungi in the species (Petrini 1986).However further study from various localities mustbe done to prove this.Table 3. Characterisation of endophytic species in Psilotum nudum    Macroscopic Characteristics            Microscopic CharacteristicsParts n Colony Colour Micelium Hypha MiceliumEndophytecolour beside texture structure structure Spore structureidentificationcolonyApex 1 LY Nill Pd None None None Noneaerial (aa)Below 1 W WDBS Pd As As Conidiophore aerial round, Aspergillus nigerapex conidia forming chain structureaerial 2 DB DB Ct As As Conidiophore aerial round Aspergillus(baa) compact column. terreus3 W WDBS Pd As As Conidiophore aerial round, Aspergillus nigerconidia forming chain structure4 W W Ct S S Conidiophore geniculate, Bipolaris sp.conidia fusoid-shaped andconsists of 5 cells.5 W W Ct S S Arthroconidia unicellular and Coccidioidesoval-shaped has large diameter. immitisMiddle 1 W W Ct S S Ovoid shaped arthroconidia. Paracoccidioidesaerial brasiliensis(ma) 2 W W Ct S S Ovoid shaped arthroconidia. Paracoccidioidesbrasiliensis3 W W Ct S S Conidiophore simple structured, Verticillium sp.single-celled conidia andovoid shaped.4 W W Ct S S Conidiophore simple structured, Verticillium sp.single-celled conidia andovoid shaped.Bottom 1 W W Ct As As Sterile mycelium structure. Deuteromycotaaerial 2 W W Ct As As Sterile mycelium structure. Deuteromycota(ba) 3 W Gy Ct As As Conidiophore simple structured, Verticillium sp.single-celled conidia andovoid shaped.4 Gn W Ct As As Conidia are round and chain. Aspergillus flavusRhizoid 1 W W Ct S S Conidiophore branched, and Scedosporium(rh) unicellular conidia obovoid apiospermumshaped.2 W DGy Ct As As Sterile mycelium structure DeuteromycotaAmmendation: LY=Light yellow, W=White, WDBS=White and dark brown spot, DB=Dark Brown, Gn=Green, Gy=Grey, Pd=Powdery, Ct=Cottony,As=Aseptate, S=Septate.ANATOMY OF SYMBIOTIC FUNGAL ENDOPHYTES IN Psilotum nudum (L.) P. Beauv 15Fig. 2. Anatomical characteristics of Psilotum nudum parts with the presence of endophytes.(A, B) Cross section of apex aerial without endophytes. (C, D, E, F) Cross section of stelesurrounded with endodermis cell layer. (G, H) Cross section of baa and ma with hyphaecoils. (I) Cross section of ba with hyphae and hyphae coils. (J) Cross section of rh withhyphae. hc, hyphae coil: h, hyphae.16 ANATOMY OF SYMBIOTIC FUNGAL ENDOPHYTES IN Psilotum nudum (L.) P. BeauvFig. 3. Fungal isolates (A) Macroscopic observation on PDA agar. (B) Microcopic observation with 400x magnification.ANATOMY OF SYMBIOTIC FUNGAL ENDOPHYTES IN Psilotum nudum (L.) P. Beauv 17Fungal Isolates CharacteristicsFungal endophyte isolates were successfullyidentified using macroscopic and microscopicobservations (Table 2, Figure 3). The identificationswere based on reference book of The Identificationof Fungi and Identifying Filamentous Fungi. Allfungal isolates have their own unique colony colorswhich can be classified into different group. Elevencolonies were successfully identified andcategorized based on parts of the species whereAspergillus niger, Aspergillus terreus, Coccidioidesimmitis and Bipolaris sp. were present on baa parts,Paracoccidioides brasiliensis and Verticillium sp.present on ma part, Aspergillus flavus, Verticilliumsp. and Deuteromycota class was found on ba,Scedosporium apiospermum and isolates fromDeuteromycota class was observed in rh parts.CONCLUSIONSThis study proved that fungal endophytecolonization assist the plant cells adaptation in theprotection against biotic or abiotic stress. These leadto beneficial properties of fungal endophytes eitherin the increasing of plant development or adaptingplant to unsuitable growth conditions. Hence,symbiotic support from fungal endophyte gave planta long life time span.ACKNOWLEDGEMENTSWe wish to thank the staff of Taman Paku PakisUKM for contributing samples for the study.Deepest thanks are also to Mohamad Ruzi AbdulRahman for guidance in the field. This research wassupported by Grant DPP-2015-144 and DPP-2015-077.REFERENCESArnold, A.E., Mejia, L.C., Kyllo, D., Rojas, E.I.,Maynard, Z., Robbins, N. & Herre, E.A. 2003.Fungal endophytes limit pathogen damage in atropical tree. In: Proceedings of the NationalAcademy of Sciences, 100: 15649-15654.Bhandari, N.N. & Mukherji, R.G. 1993. TheHaustorium. Research Studies Press, New York.122 pp.Dickison, W.C. 2000. Integrative Plant Anatomy.Harcout Academic Press, San Diego. 146 pp.Elizabeth, M.L. 1972. Fundamentals of the Fungi.Prentice–Hall Incorporated, New York. 78 pp.Gehring, C. & Bennett, A. 2009. Mycorrhizalfungal-plant-insect interactions: The importanceof the community approach. 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Effects of dietary flavonoids onappoptatic pathways related to cancerchemoprevention. The Journal of NutritionalBiochemistry, 18: 427-442.18 ANATOMY OF SYMBIOTIC FUNGAL ENDOPHYTES IN Psilotum nudum (L.) P. BeauvRodriguez, R.J., White Jr., J.F., Arnold, A.E. &Redman, R.S. 2008. Fungal endophytes :Diversity and functional roles. New Phytologist,182(2): 314-330.Saas, J.E. 1958. Botanical Microtechnique. 3rd Ed.Ames: Iowa State University Press.Vega, C., Ortiz, P.L., Arista, M. & Talavera, S. 2007.The endophytic system of MediterraneanCytinus (Cytinaceae) developing on five hostCistaceae species. Annals of Botany, 100: 1209-1217.Vega, C., Arista, M., Ortiz, P.L. & Talavera, S. 2010.Anatomical relations among endophytic holo-parasitic angiosperms, autotrophic host plantsand mychorrizal fungi: A novel tripartiteinteraction. American Journal of Botany, 97(5):730-737.Yamazaki, Y., Dae-yeon, S., Sitthithaworn, W.,Ishiguro, K., Kobayashi, W., Shibuya, M.,Yutaka, E. & Sankawa, U. 2001. Diversechalcone synthase superfamily enzymes fromthe most primitive vascular plant, Psilotumnudum. Planta, 214: 75-84.

Anatomy of symbiotic fungal endophytes in

Psilotum nudum (L.) P. Beauv

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Anatomy of symbiotic fungal endophytes in Psilotum nudum (L.) P. Beauv

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