Characterization, Antimicrobial and Anti-fungal Activity

Journal of Society & Technology 5:162-172 (2015)

Characterization, Antimicrobial and Anti-fungalActivity of Endophytic Fungi Associated withGelidiella acerosa (Forsskal) Feldmann and G.Hamel (1934) from the Intertidal Area of Silago,Southern Leyte, Philippines

Berna Lou L. Aba* and Neil Michael M. AlmosaVisayas State University, Visca, Baybay City, Leyte, Philippines

Abstract

Endophytic fungi that live inside plant tissues are known to protect their hosts againstpathogens.Thus this study was conducted to isolate, characterize and identify endophyticfungi from the red algae Gelidiella acerosa in the intertidal area of Balagawan, Silago, SouthernLeyte and evaluate their antibacterial and antifungal activities. Endophytic fungi were isolatedand cultured in Potato dextrose agar (PDA), characterized, identified up to division level onlyand were subjected to antibacterial and antifungal assays respectively. Eighteen endophyticfungi were isolated from Gelidiella acerosa from three sampling sites during a two samplingperiod. Results showed sixteen fungal isolates belonging to division Deuteromycota and onespecies each to divisions Ascomycota and Zygomycota. Escherichia coli and Bacillus subtiliswere used in the antimicrobial assay as test organisms against the fungal isolates and Penicillinas the positive control. For the antifungal assay, Helminthosporium maydis and Sclerotiumrolfsii (fungal pathogens) were used as the test organisms and Trichoderma harzianum as thepositive control. Antibacterial assay results showed no significant difference between isolates(FI1 to FI18) for both E. coli and B. subtilis. Antifungal assay between isolates also showedno significant difference against the two pathogens tested. Moreover, identification of isolateto species level and verification of antibacterial and antifungal activity is recommended.

Keywords: endophytic fungi, antibacterial assay, antifungal assay, Gelidiella acerosa

Introduction

Endophytic fungi are a group of fungi thatcolonize living, internal tissues of plants eitherfor a short or prolonged period withoutcausing any immediate or negative effects(Hirsch and Braun, 1992) on the plant.Recent studies have revealed the ubiquity ofthese fungi, with an estimate of at least 1million species of endophytic fungi residing inplants (Dreyfuss and Chapela, 1994) and evenin lichens (Li et al., 2007). They produce anextraordinary diversity of extrolites; some ofwhich have therapeutic value as novelantibiotics or anticancer chemicals(Gunatilaka 2006, Suryanarayanan et al.,2009). They have not been studied, however,

to any extent for their enzyme potential(Govindarajulu et al., 2011). Recently, theyhave received considerable attention afterthey were found to protect their host againstinsect pests, pathogens and even domesticherbivores (Shiomi et al., 2006). A wide rangeof parasitic and saprophytic fungi have beenreported from marine algae (living in thetissue, as epiphytes, rather than within), butfew studies have investigated the endophytesof marine algae (Jones et al., 2008). Of thestudies performed on marine endophytes,most have studied algae from temperateregions such as the German coast of theNorth Sea (Zuccaro et al., 2008), but not yetin other countries. Gelidiella acerosa(Gelidiales, Rhodophyta) is a common

*Correspondence: [email protected]

Journal of Society & Technology Aba & Almosa

component of seaweed communities in thePhilippine coastal waters. The speciesproduces high quality agar that is used in thelocal and world agar industry along withGracilaria, Gelidium and Pterocladia. Becauseof this value, cultivation and technology forGelidiella acerosa is being developed. Hence,this species of algae represents a potentialsource for the investigation of endophyticfungi’s antimicrobial and antifungal activity.

Materials and Methods

Sampling Site

Figure 1: Map of Silago, Southern Leyteshowing the sampling sites (Google Maps).

Description of Sampling Site

The sampling site is located at the intertidalarea of Brgy. Balagawan, Silago SouthernLeyte, Marine Sanctuary at 10o28’26.49”N &125o11’28.87” E (Google Earth, 2012). Themarine resources of Silago identified in theCLUP include 1,232 hectares of coral reefs,128 hectares of mangroves as well as sea grassareas, tidal flats, estuaries and rock cliffs. Theintertidal area of the sanctuary is rocky that isa good substrate for seaweeds like Sargassumsp. and Gelidiella acerosa. Three samplingsites were chosen, which are located at theleft side of the marine sanctuary where thereis an abundance of seaweeds. The MarineSanctuary is well established and watched bythe surrounding community, where strict

compliance of the sanctuary rules wasobserved.

Collection of Samples

In sampling site 1, a line transect was laid onthe left side of the shore which is a fewmeters away from the mouth of the river. Site2 was 15 meters away from Site 1, located inbetween Site 1 and 3. Sampling site 3 on theother hand was situated at the right side,facing the shoreline. Each transect wasdivided into three subsites where in 10g ofGelidiella acerosa samples were collected andplaced in sterile plastic bags. Immediatelyafter the collection of samples, the sampleswere brought to the DBS Laboratory andprocessed after the 4-5 hour ride from thesite. Collection of samples was done in Marchand December, 2011. Physico-chemicalparameters (water depth, temp, salinity, andpH )were also noted.

Experimental Design

Fungal isolates from each transect wascategorized as subsamples. Evaluation andcomparison of the antimicrobial activity of theendophytic fungi species was done inCompletely Randomized Design (CRD) usingthe fungal isolates (FI1-FI18) as thetreatments for the data on antibacterial andantifungal assay. Three replications for everytreatment were employed.

Isolation of Endophytic Fungi

The algal parts were rinsed gently in steriledistilled water to remove debris and otheradhering particles. After proper washing,algae were cut using sterile scissors into smallpieces (3-4 mm in diameter and 0.5-1 cm inlength). Isolation of endophytic fungi wasdone through surface sterilization (Petrini etal., 1986). It was done using sodiumhypochlorite (NaOCL) and ethanol at 30%and 75% concentrations, respectively. Eachset of algal segments were treated withNaOCL for 3-10 minutes the transferred to

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75% ethanol for 30 seconds. Then, each ofthe segments was rinsed three times withsterile distilled water. The algal pieces wereblotted on sterile blotting paper. In eachsterile petri dish containing previouslyhardened sterile Potato 7 Dextrose Agar(PDA), 5 segments were placed. The plateswere sealed with parafilm and incubated at27oC ± 2oC for 2-3 weeks. Most of the fungalgrowth was expected to start within 4 days ofinoculation. The incubation period for eachfungus was recorded. The day of first visiblegrowth was recorded from the date of platingand was considered as the incubation periodfor growth. Isolation of fungal growth fromthe master plates were done by the transfer ofhyphal tips to fresh sterile PDA plates withoutaddition of antibiotics to obtain pure culturesfor identification and characterization.

Identification and Characterizationof Endophytic Fungi Isolated

For preliminary identification, morphologicalexamination of the colony shape, color, sporeformation as well as the texture of fungalgrowth was done. In order to observe fungalmorphology, slide mounts was made usinglactophenol cotton blue as the mountingmedium. This was poured out at the sides ofthe cover slip with the use of a dropper untilit was absorbed by the fungi. The preparationwith the cover slip was carefully heated overthe flame in a moderate temperature for theagar cube to melt and to exclude air bubbles.The cover slip was pressed gently with adissecting needle. In order to preserve theoverall morphology and have temporarymounts of isolated fungi, the sides of thecover slip was sealed using a colorless nailpolish. The mounted slide was viewed underan electric microscope at high power objective(HPO) and oil immersion objective (OIO) formorphological characterization. The mountedslides was placed in a wooden box andlabelled properly for future identificationpurposes (Bitacura, 2010).

Antimicrobial Activity Tests

Antibacterial assay

Qualitative evaluation of the antibacterialactivity of the fungal isolates was donethrough a disk diffusion assay by Kirby-Bauer(1961) with slight modification of theprotocol. Test organisms used were Bacillussp. (Gram-Positive bacteria) and E. coli(Gram-Negative bacteria). Antibacterialagents from fungal isolates were prepared byculturing the isolates in Potato DextroseBroth (PDB) for 2 weeks without shaking,and 100 ppm final concentration of Penicillinwas used as the positive control for theexperiment. Paper disks were prepared (5mmin diameter) and were sterilized. Sterile paperdisks were immersed in the beaker containingpositive control (Penicillin). The same wasdone for each of the 2- week culture of Fungalisolates on the PDB. Meanwhile, testorganisms were grown in nutrient broth for24-36 hours and 20 µL were inoculated intothe plates by spread plate method. On thesurface of nutrient agar plates with E. coliand plates with B. subtilis, the disks soakedon the different fungal isolates were placedequidistantly from each other. Plates werethen incubated for 48 hours at roomtemperature. Zones of inhibition evident onthe plates were measure in mm from thecenter of disk to the edge of bacterial growth.

Antifungal Assay

Antifungal potentials of the fungal isolateswere evaluated and compared throughantagonism test using the plant pathogenicfungi Helminthosporium maydis andSclerotium rolfsii. Two week old fungalisolates cultured on plates of PDA were usedas the source of antifungal agents. While aculture of Trichoderma harzianum was usedas the positive control for the experiment.PDA plates at 15 ml/plate, were prepared. A5 mm diameter cork borer was then used tocut and transfer isolates (agar plugs), to theculture plates with Helminthosporium maydis,

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Sclerotium rolfsii and Trichoderma harzianum.Three replicates were employed per treatment(fungal isolates). Agar plugs of the testsorganisms and the fungal isolates were placedat opposite ends and plates were sealed withparafilm and incubated at room temperaturefor one week. Extent of inhibition wasdetermined in each plate using the scaledeveloped by Bell et al. (1982) as follows:

1 Antagonists completely overgrew thepathogen and covered the entire medium.

2 Antagonists overgrew at least 2/3 of themedium surface.

3 Antagonists and pathogen each colonizedapproximately 1

2 of the medium surface(more than 1/3 and less than 2/3).

4 Pathogen colonized at least 2/3 of themedium surface and appeared towithstand encroachment by antagonists

5 Pathogen completely overgrew theantagonists and occupied the entiremedium surface.

Statistical Analysis

Correlation of the physico-chemicalparameters to the colonization frequencies ofthe fungal isolates was done using Pearson’sCorrelation Analysis. While data onantimicrobial activity tests was analyzedfollowing the Analysis of Variance (ANOVA)for a Completely Randomized Design (CRD)to test the significance of the treatmentsconsidered.

Results and Discussion

Endophytic Fungi Isolated fromGelidiella acerosa

A total of eighteen fungal isolates wererecorded from the G. acerosa samples, ninefrom each sampling period as shown in Table1. Based on the analysis of the morphologicaland microscopic characteristics of the isolated

fungi, it appears to belong to divisionsDeuteromycetes (16), Ascomycetes(1) andZygomycetes respectively. According to Jonesand Alias, 1997; Hyde and Lee, 1995;Kohlmeyer and Kohlmeyer, (1979), the mostabundant source of marine fungi in thecoastal environment is macrophyte detritus,such as decaying magrove leaves, sea grasses,macroalgae and wood. These lignicolousmarine fungi mostly belong to theAscomycetes, Basidiomycetes andDeuteromycetes. The shoreline of BalagawanMarine Sanctuary is abundant of decayingdrift wood, and mangrove leaves and this mayhave contributed to the abundance of isolatedendophytic fungi in the area.

Results also showed very slight difference inthe fungal isolates collected between twosampling periods which indicates no diversity.The first group of isolates (Isolates FI1-FI8)were collected at 0.9-0.15 m depth with atemperature range of 27-28oC while that ofthe second group (Isolates FI9-FI18) weresampled at 0.14-0.18 m depth at temperaturerange of 30-31.8oC. Isolate FI7 that belong toAscomycete was collected during the firstsampling but not found in the second, whileisolate FI18, belonging to Zygomycetes wasrecorded during the second sampling but notin the first, along with the Deuteromycetesthat were collected in both sampling periods.According to Booth and Kenkel (1986) one ofthe major factors that govern fungal diversityis geographical distribution and thetemperature of the sea in the case of marinefungi. Hughes (1974) was the first to publishdistribution maps of marine fungi and dividethe oceans into zones based on their averagetemperature range over the year. This has ledto a number of maps showing the worlddistribution of marine fungi (Kohlmeyer, 1983;Hyde and Lee, 1995). While these maps help,at a glance, to indicate trends in thegeographical distribution of marine fungi, theyare limited to the extent that vast areas havenot been sampled.

Furthermore, species diversity can be low inthe intertidal due to the prevailing

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Table 1: Characteristics of Isolated Endophytic Fungi

Isolates Division Characteristics

Isolate FI1, FI4, FI5, FI6,FI8, FI12, FI13, FI14,FI15, FI16, FI2, FI10,FI11, FI3, FI9 and FI14

Deuteromycetes

Colony color ; white, pinkish, light brown, dirtywhite, and black; none spore former, smooth torough texture of colony; thousands of septatehyphae, sexual structures or morphology notobserved.

Isolate FI7 *a AscomycetesColony is yellowish in color and appears to havea rough cottony texture. Non-motile spores notevident, do not form asci or ascopors

Isolate FI18 *b Zygomycetes

Colony is white in color and appears to have aslight cottony texture. This fungal isolate is nospore formed hyphae may be coenocytic, formingsepta only where gametes are formed

*a - found in first sampling only*b - found in second sampling only

environmental conditions of desiccation,varying salinity and temperature and exposureto UV light. As in the case of the shoreline ofBalagawan Marine Sanctuary, salinity rangesbetween 33-35% and water current rangesfrom 6-10m/s. Also, other factors liketyphoons and changing seasons affects thedepths of the water pools where the Gelidiellaacerosa are found along with change inoptical density and pH of the water. Many ofthe Ascomycetes, however, survive theseconditions because its developing asci areembedded in mucilage in the centrum or theascomata are immersed deeply within the host(Au et al., 1999). Nevertheless in this study,only one isolate was recorded to be underAscomycete, and more of Deuteromycetes (16isolates) which are known to have no definitesexual stages. Accordingly, this is a similartrend that is observed by Frohlich and Hyde(1999) where the majority of the endophyticfungi they collected belonged to these twodivisions. Hence, it could be concluded thatcolonization and diversity of marine fungi(endophytic or not) depends on geographicalregions, extent of salinity, kind of substrates,and position of intertidal region, nature offloor, pH and oceanic region.

Also, according to Carroll, (1988), majorityof fungal endophytes are represented withinthe division Ascomycota isolated from brown

algae, and from green algae (Suryanarayananet al. 2010). However, results of this studyshowed only one fungal endophyte isolatedunder Ascomycetes. Possibly this is becauseG. acerosa belongs to the red algae andprobably favours for endophytic fungibelonging to Deuteromycetes. Aspergillus sp.under division Ascomycota was found to bethe dominant genus of endophyte speciesisolated from algae, with no restrictionregarding the host alga (i.e. from red, brownor green algal species) (Suryanarayanan et al.2010). However more research needs to bedone to see this trend among endophyticfungi (Suryanarayanan et al. 2010). As in thecase of this study, results showed moreisolated endophytes belonging to divisionDeuteromycota than Ascomycota.

Antibacterial Assay

Results of the antibacterial assay from theisolated fungi shows no significant differenceagainst E. coli and B. subtilis (p >0.05)(Figure 6 and 7). Among the first group ofisolates, it was recorded that FI9 shows thehighest zone of inhibition against E. coli whileisolates FI1, FI3, and FI7 (the onlyAscomycete among the samples) shows nozone of inhibition. Yet, although no zone ofinhibition was noted, it was observed that thedisks of this fungal isolates were not colonized

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Table 2: Average zones of inhibition (mm) of fungal isolates from Gelidiella acerosa andpenicillin against Echerichia coli and Bacillus subtilis

Fungal IsolateZones of inhibition (mm)

Echerichia coli Bacillus subtilis

FI1 0 0FI2 2 3FI3 0 0FI4 1 2FI5 2 2FI6 2 2FI7 0 0FI8 1 1FI9 3 1FI10 2 2FI11 2 2FI12 2 1F113 2 1FI14 3 2F115 2 2FI16 0 0FI17 2 3FI18 2 2100 ppm Penicillin (positive control) 12 6

by E. coli. This means that these isolatescould possibly inhibit the bacteria fromcolonizing but possibly, the antimicrobialproperty needs to be purified so as to have itshighest antimicrobial potential. Moreover,when antibacterial activity for all the isolateswas compared to the control Penicillin(100ppm), there was a highly significant difference(p <0.05) observed for both E. coli and B.subtilis.

According to Mao et al., (2006),antibacterial potential could be possibly dueto the expected different modes of action, thelevel of isolate inoculation and activity of theindividual biochemical constituent of therespective isolates. Furthermore, differentisolates produce a host of different number ofcompounds/metabolites where thesemetabolites also have different molecularweights causing the difference in yields(Newman et al., 2003). In the case of thisqualitative study, it is important to note thatthe degree of inhibition of the isolates against

the test organisms may be also attributed tothe purity of the antimicrobial agent, where inthis case was not purified and identified.However some of the test isolates had fairlyhigh activity as well. It is possible that therewere novel compounds from the test isolatesand could be promising agents to replacedrugs which pathogens have developedresistance through time. However furtherpurification of such compounds and chemicalanalysis is required to determine this.

Furthermore, on the colonization ability ofthe isolates, results show that fungal isolateshave an antagonistic potential against H.maydis and S. rolfsii since it colonized 1/2, toabout 2/3, of the medium surface based onthe scale developed by Bell et al., (1982).However these fungal isolates have lowerextent of antagonism against the pathogensas compared to Trichoderma harzianum(positive control). Among the isolates and thecontrol, no significant difference was observed.

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Images of Fungal Isolates onFirst Sampling

Figure 2: Fungal Isolates FI1, FI2 and FI3.Two week old colony of endophytic fungi thatis under Deuteromycetes (A (FI1) ,B (FI2)and C (FI3) ). Fungal hyphae shown at OIOat 1000x magnification.

Figure 3: Fungal Isolate FI7. Two weekold colony of endophytic fungi that is underAscomycetes. Hyphae of isolate G (FI7)shown at OIO at 1000x magnification.

Yet, the observed antifungal potential ofthe isolates may be attributed to thebiologically active compounds in the fungal

isolates which according to Jones et al.,(2008) are significant source of new drugs.Although the colonization frequencies of theisolates were not higher than the control andno significant differences were observed, it isstill possible that they have bioactivecomponents are more potent than the controlwhen purified and tested to other pathogensaside from the pathogens used in this study.Also, the different values obtained with thedifferent fungal phytopathogens usedindicated that the direct interaction betweenendophytic fungus and pathogen was complexand may exhibit species-specific antagonismas previously described by Arnold et al.,(2000). Moreover, Jones et al., (2008) andKjer et al.,(2010) said that recently, moststudies have focused on marine endophytesdespite being much less common, has led tothe discovery of new antimicrobial compoundswhich may be an untapped source of newchemical diversity.

Images of Fungal Isolates onSecond Sampling

Figure 4: Fungal Isolate FI10 and FI15.Two week old colony of endophytic fungi thatis under Deuteromycetes (J (FI10) and O(FI15) ). Hyphae of isolate shown at OIOat 1000x magnification.

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Figure 5: Fungal Isolate FI18. Two weekold colony of endophytic fungi that is underZygomycetes (R(FI18)). Hyphae of isolateat OIO at 1000x magnification

According to Mao et al., (2006),antibacterial potential could be possibly dueto the expected different modes of action, thelevel of isolate inoculation and activity of theindividual biochemical constituent of therespective isolates. Furthermore, differentisolates produce a host of different number ofcompounds/metabolites where thesemetabolites also have different molecularweights causing the difference in yields(Newman et al., 2003). In the case of thisqualitative study, it is important to note thatthe degree of inhibition of the isolates againstthe test organisms may be also attributed tothe purity of the antimicrobial agent, where inthis case was not purified and identified.However some of the test isolates had fairlyhigh activity as well. It is possible that therewere novel compounds from the test isolatesand could be promising agents to replacedrugs which pathogens have developedresistance through time. However furtherpurification of such compounds and chemicalanalysis is required to determine this.

Furthermore, on the colonization ability ofthe isolates, results show that fungal isolateshave an antagonistic potential against H.maydis and S. rolfsii since it colonized 1/2, toabout 2/3, of the medium surface based onthe scale developed by Bell et al., (1982).However, these fungal isolates have lowerextent of antagonism against the pathogens

as compared to Trichoderma harzianum(positive control). Among the isolates and thecontrol, no significant difference was observed.Yet, the observed antifungal potential of theisolates may be attributed to the biologicallyactive compounds in the fungal isolates whichaccording to Jones et al., (2008) aresignificant source of new drugs. Although thecolonization frequencies of the isolates werenot higher than the control and no significantdifferences were observed, it is still possiblethat they have bioactive components are morepotent than the control when purified andtested to other pathogens aside from thepathogens used in this study. Also, thedifferent values obtained with the differentfungal phytopathogens used indicated that thedirect interaction between endophytic fungusand pathogen was complex and may exhibitspecies-specific antagonism as previouslydescribed by Arnold et al., (2000). Moreover,Jones et al., (2008) and Kjer et al.,(2010)said that recently, most studies have focusedon marine endophytes despite being much lesscommon, has led to the discovery of newantimicrobial compounds which may be anuntapped source of new chemical diversity.

Summary, Conclusion andRecommendation

Gelidiella aerosa samples were taken in thethree different points of the intertidal area inBrgy. Balagawan, Silago, Southern Leyteduring the month of March and December2012. Laboratory analyses were conducted atthe Microbiology laboratory in theDepartment of Biological Sciences, VisayasState University, Baybay City, Leyte. Thisstudy aimed to isolate, characterize andidentify the endophytic fungi associated withGelidiella aerosa to determine theantibacterial and antifungal activities of theendophytic fungi collected. Endophytic fungiisolates were grown in Potato Dextrose Agar(PDA). The colonies that grew were isolatedinto pure culture through sub culture and

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microscopic methods. Preliminaryidentification, microscopic and colonialcharacterization was done. The identificationof isolated 15 endophytic fungi were verifiedand confirmed up to division level only by theDepartment of Pest Management, VisayasState University. Antibacterial assay using E.coli and B. subtilis as test organisms and 100ppm final concentration of Penicillin as thepositive control were prepared. Pure cultureswere then subjected to an in-vitro antifungalassay using Helminthosporium maydis andSclerotium rolfsii as test organisms andTrichoderma harzianum as the positivecontrol.

A total of eighteen endophytic fungi wereisolated from the first and second samplingperiod. Sixteen of the isolates belong to ClassDeuteromycetes and one to ClassAscomycetes and Zygomycetes. There was anantibacterial and fungal colonization observedamong the isolates however were found to benot significantly different.

The fungal isolates collected displayedpotential bioactive compound activity,however, it is recommended to further thisstudy by purifying and further testing of thebioactive compounds (extrolites) that may bepresent in these isolates. Also, molecularmethods of identifying such unknownorganisms are highly recommended.

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