ISSN: 1412-033X (printed edition) ISSN: 2085-4722...

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic)

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ISSN: 1412-033X (printed edition)

2085-4722 (electronic)

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B I O D I V E R S I T A S ISSN: 1412-033X (printed edition)Volume 10, Number 4, October 2009 ISSN: 2085-4722 (electronic)Pages: 163-167 DOI: 10.13057/biodiv/d100401

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Comparison Analysis of Genetic Diversity of IndonesianMangosteens (Garcinia mangostana L.) and Related Species by

Means Isozymes and AFLP Markers

SOBIR1,, SOALOON SINAGA2,, ROEDHY POERWANTO1, RISMITASARI3, RUDY LUKMAN41Departement of Agronomy and Horticulture, Institut Pertanian Bogor (IPB), Bogor 16680

2Kopertis Wilayah III, Jakarta 136403Centre for Plant Conservation Bogor Botanic Gardens, Indonesian Institutes of Sciences (LIPI), Bogor 16122

4Bisi International & SEAMEO BIOTROP, Bogor 16720

Received: 2nd March 2009. Accepted: 26th June 2009.

ABSTRACT

Mangosteen (Garcinia mangostana) belongs to a large genus of Garcinia that native in South East Asia, as well asIndonesia, and in order evaluate genetics diversity of mangosteen and their close relatives, we employed isoenzyme andAFLP marker on 13 accessions of mangosteen and their close relatives. Isoenzyme marker using four enzyme systemsproduced 25 bands and 88% out of them were polymorphic and elucidate genetic variability at similarity level rangedbetween 0.38-0.89. AFLP markers with three primer system produced 220 polymorphic bands and revealed geneticvariability at similarity level ranged between 0.38-0.89 successfully produced high polymorphism bands and elucidatesgenetic variability at similarity coefficient ranged between 0.21-0.77. Both markers exhibited similar clustering pattern, andgroup successfully G. mangostana accessions in one clustering group. Furthermore G. malaccensis and G. porrectaconsistently showed closer genetic relationship to G. mangostana clustering group in both markers, in comparison to G.hombroniana, which implies the assumption they may be the progenitor of G. mangostana, and should be reviewed with moreaccurate data.

2009 Biodiversitas, Journal of Biological Diversity

Key words: genetic diversity, mangosteen, isozymes, AFLP.

INTRODUCTION

Mangosteen (Garcinia mangostana L.) belongs tofamily Guttiferae, genus Garcinia (Verheij, 1991).Garcinia is a large genus that consists of about 400species, and originated from East India, Malay Peninsulaand South East Asia, as well as Indonesia (Campbell1966). Based on morphological and cytologicalstudies, Yaacob and Tindall (1995) suggested thatmangosteen originated from South East Asia;subsequently Almeyda and Martin (1976) proposedthat mangosteen is an inhabitant Indonesian fruit.

Some species of Garcinia, including G.mangostana produce fruit without pollination, thephenomenon is referred to as agamospermy, which isthe production of seed without fusion of gametes(Koltunow et al., 1995; Thomas 1997). The process ofembryo formation in G. mangostana was first studied

by Treub (1911) who reported that the earlydevelopment of woodiness in the endocarp soon afteranthesis made the observation of embryodevelopment difficult (Tixier, 1955). However, Lan(1989) provided a detailed account of mangosteenembryology and reported that the embryo of G.mangostana is derived from tissue of integumentinstead of from the egg.

An understanding of genetic diversity and itsphylogeny among cultivated plant accessionsignificantly influence on the quality increase and theresults, and it also improves the management ofgermplasm conservation (Roldan-Ruiz et al., 2001).Plant genetic improvement highly depends on theavailable genetic resources. Wide genetic diversitywill give higher opportunity in the selection process ofthe best characters. Some research on the geneticdiversity using some markers could explain thephylogeny within and among population (Fajardo etal., 2002; Hurtado et al., 2002; McGregor et al., 2002).

Genetic variability analysis can be done by usingmany manner of markers, such as morphology(Talhinhas et al., 2006), isoenzymes (Ayana et al.,2001), and molecular markers (Assefa et al., 2003;Cavagnaro et al., 2006), such as AFLP marker (Vos

BIODIVERSITAS Vol. 10, No. 4, October 2009, pp. 163-168164

et al., 1995). Recently, due to burgeoning inbiotechnological technique, the molecular markershave been widely used to elucidate geneticinformation in the molecular level (Roy et al., 2006).

Each marker system has the advantages anddisadvantages, so that the assessment of themarkers system is an important step to decide themost suitable marker regarding to research purpose.The comparison of several markers has been donewith comparative study of some molecular markerswith PCR base such as Palombi and Damiano (2002)which compared RAPD and SSR markers to detectgenetic variability of kiwi plant, Ferdinandez andCoulman (2002), compared the efficiency of RAPD,SSR, and AFLP to identify plant genotypes. Saker etal. (2005), has used different markers to characterizethe barley.

The study is aimed to distinguish the advantages ofisoenzyme and AFLP markers in elucidating geneticvariability and phylogenetic relationships among themangosteen (Garcinia mangostana L.) and the closerelatives, and to study the suitable molecular todevelop specific molecular markers in characteri-zation of mangosteen and its close relatives.

MATERIALS AND METHODS

Plant materialThis research was conducted in the laboratory of

Biotechnology and Tree Breeding BIOTROP Bogor,Molecular Laboratory and Plant Biology the ResearchCenter for Biological Resources and BiotechnologyIPB Bogor, and Laboratory of Tropical Fruit ResearchCenter IPB Bogor. Thirteen (13) leaf samples ofmangosteen and its close relatives were collectedfrom several locations in Indonesia, namely:Pandeglang (Banten), Sukabumi, Purwakarta (WestJava), Ponorogo (East Java), Lampung Regency,Palangkaraya (Central Kalimantan), Kendari (SouthEast Sulawesi), Ambon (Maluku), G. rigida, G.hombroniana, and G. celebica (Bogor BotanicalGardens), and G. malaccensis, G. porrecta, and G.benthami (Mekarsari Tourism Park Bogor).

Isoenzymes analysisThirteen fresh samples were taken for isozyme

analysis following Soltis and Soltis (1989). Theenzymes analyzed are peroxidase (PER),phosphatase acid (ACP), malic dehydrogenase(MDH), and esterase (EST). The separation ofisoenzyme bands was done with electrophoresis byusing agarose gel with concentration of 10% for 4hours, and 100 volt.

AFLP analysisExtraction and DNA purification

The DNA of Leaf samples were extracted for AFLPanalysis the same as for isoenzyme analysis. DNAextraction followed CTAB (Doyle and Doyle, 1987)

with some modifications. DNA concentration wastested with electrophoresis and immigrated withstandard DNA (DNA lambda) 10 and 100 ng/mL onagarose gel 1.2%.

Restriction-ligationApproximately 0.5 g genomic DNA was cut 1 unit

MseI and 5 unit EcoRI. At the same time it is ligatedwith 5 pmol EcoRI and 50 pmol MseI adaptor with 1 UT4 DNA ligase. The adaptor sequence EcoRI is 5-CTCGTAGACTGCGTACC-3, 3-CTGACGCATGGTTAA-5 and the adaptor sequenceMseI is 5-GACGATGAGTCCTGAG-3, 3-TACTCAGGACTCAT-5.

Preselective amplificationPrimers for preselective amplification are EcoRI+A

and MseI+C as homologous adaptor EcoRI and MseI,each with one additional nucleotide at 3 end. PCRreactions were carried out in reaction mix containingof 4 l restriction-ligation DNA, 2.5 pmol primer EcoRI+A and 2.5 pmol MseI primer +C, 0.4 U Taqpolymerase DNA, 0.2 mM each dNTP and 1x bufferPCR 20 L. The PCR amplification was programmedfor 20 cycles at 94 (1 second), 56C (30 seconds),and 72C (2 minutes). The PCR products 10 uL wastested w on 1.5% agarose gel. The amplifiedfragments range from 100-1500 bp.

Selective amplificationThe selective amplifications were conducted by

using primer EcoR1+ ANN and Mse1+CNN. The PCRreaction was performed using DNA pre-amplification3 L, 1 pmol primer EcoRI + ANN, 5 pmol primerMseI + CNN without labeling, 0.4 U Taq polymeraseDNA, 0.2 mM each dNTP and 1 x buffer PCR with atotal volume of 20 l. PCR reaction was programmedwith 1 cycle for 30 seconds at 94C, 30 seconds65C, 2 minutes at 72C, followed by eight cycles ofvariable annealing temperature with a decrease of1C each cycle, and terminated with 23 cycles of 1second at 94C, 30 seconds at 56C, 2 minutes at72C.

PAGE electrophoresisThe selective amplification products were

displayed using PAGE electrophoresis, andpresented as a diagram. Approximately 2 L PCRproduct mixed with 0.15 L 6-carboxy-Xrhodamin(ROX)-labeled internal standard length GeneScan-500 ROX and dye 0.85 L formamide, denaturized for3 minutes at 90C and cooled in ice. Electrophoresisusing 5% gel denaturing polyacrylamide (LongRangerTM, FMC Bioproducts) in bufferelectrophoresis 1x TBE by using ABI PrismTM 377DNA sequencer (Applied Biosystems) at 2500 V for 4hours. The raw data was obtained using ABIPRISMTM V.1.1 software. Next, the AFLP fragmentswere analyzed with GENESCANTM version 2.1(Applied Biosystems).

SOBIR et al. Isozymes and AFLP diversity of Indonesian mangosteen 165

Data analysisThe bands of the isozyme technique and AFLP

were translated into the binary data. These data wereused to arrange the genetic similarity matrix based onthe formula of Nei and Li (1979) with UPGMA(Unweighted Pair-Group Method Arithmetic) methodusing NTSYS (Numerical Taxonomy and MultivariateSystem) version 2.02 (Rolf, 1998). Genetic similaritybetween all pairs of accessions was calculatedaccording to Nei and Li (1979).

RESULTS AND DISCUSSION

Variability analysis with isozyme markerIsozymes analysis on 13 accessions of

mangosteen and their close relatives showed that thefour isoenzyme systems of esterase (EST),peroxidase (PER), acid phosphatase (ACP), andmalic dehydrogenase (MDH) produced 25 bands and22 bands (88%) out of them were polymorphic band(Table 1).

Table 1. The number of bands and polymorphism level of 5isoenzyme on 13 accessions of mangosteen and their closerelatives.

Isoenzymes BandnumberPolymorphic

bandsMonomorphic

BandEST-1 4 4 (100%) 0EST-2 3 3 (100%) 0EST-3 3 3 (100%) 0PER-1 2 2 (100%) 0PER-2 3 3 (100%) 0PER-3 1 0 (0%) 1ACP-1 1 1 (100%) 0ACP-2 3 2 (66,7%) 1MDH-1 1 0 (0%) 1MDH-2 4 4 (100%) 0

25 22 (88%) 3

Cluster analysis based on isoenzyme assayrevealed, that genetics distance among 13accessions of mangosteen and their close relativesranged between 0.38-0.89 of similarity coefficient(Figure 1). The similarity matrix correlation valueMxComp r = 0.902 indicated that the dendrogramproduced with goodness of fit highly compatible whichdepict the cluster (Rolf, 1998). Presentationaccumulation of the three main first components onthe 13 accessions of mangosteen and its relativesrepresent 63,5% genetic diversity that explained by25 isozyme characters, and 70% genetic diversitywas obtained from accumulation of four maincomponents.

Subsequently, isozyme analysis showed thatmangosteen accessions and G malaccensis areclustered at 0.68 of similarity coefficient (32%)separated to other close relatives (Figure 1). Thegenetic diversity resulted from similarity analysis wasrelatively high for the obligate apomictic compared toTaraxacum (19%) (Ford and Richards, 1985).

Variation in apomictic plants occurred faster inmutation (Hughes and Richards, 1985). This resultsindicated that isozyme analysis successfully groupedmangosteen out of their close relatives, and Gmalaccensis closer to mangosteen than other closerelatives. However, further analysis showed that G.porrecta has closer genetic relationship to G.mangostana clustering group at 0.61 of similaritycoefficient, compare to G. hombroniana which isassumed as another progenitor of mangosteen(Richards, 1990), indicated that isozyme assay notyet confirmed G. hombroniana as G mangostanaprogenitor.

Koefisien kemiripan0.38 0.51 0.63 0.76 0.89

Lampung

G.malaccensis_b

Kalteng

Kusu-kusu

Banten

Wanayasa

Sukabumi

Ponorogo

G.porrecta

G.rigida

G.hombroniana

G.celebica

G.benthami

Figure 1. Dendogram of 13 accessions based on isozymemarker.

Variability analysis with AFLPAFLP analysis on 13 accessions of mangosteen

and their close relatives using three primercombinations of ACC_CAG, ACT_CAA andACT_CAC produced 220 polymorphic bands at bandsize ranged between 50-500 bp. The number ofbands resulted from each primer combination variedbetween 19-94 bands or at average 73.3 bands foreach primer combination. The primer combination ofACT_CAA produced the highest number ofpolymorphic (94 bands) followed by primercombination of ACT_CAA 70 bands and primerACC_CAG 56 bands (Table 2).

Cluster analysis results based on AFLP markers,showed that genetics distance among 13 accessionsof mangosteen and their close relatives ranged atbetween 0.21-0.77 (Figure 2). Based on the AFLPdendrogram, this hypothesis can be accepted. Withvalue r = 0.977, meaning that the dendrogramresulted with goodness of fit very suitable to depictthe grouping. Principle component analysis indicatedthat the three main first components represented47.2% genetic diversity, and 70% genetic diversity of612 characters was obtained from accumulation of sixmain components.


Table 2. The number of bands and polymorphism of 3 pairsof primer AFLP on 13 accessions of mangosteen and closerelatives.

Primer AFLP Band number Polymorphic bandsACC_CAG 94 100%ACT_CAA 70 100%ACT_CAC 56 100%Total 220 100%

Koefisien kemiripan0.21 0.35 0.49 0.63 0.77

Lampung

G.porrecta

Kalteng

Sukabumi

Ponorogo

Banten

Wanayasa

G.malaccensis_b

Kusu-kusu

G.hombroniana

G.benthami

G.celebica

G.rigida

Figure 2. Dendogram of 13 accessions based on AFLP marker.

Further analysis on dendrogram constructed fromAFLP marker indicated that mangosteen accessionsclustered in one group with G. porrecta, separatedwith other close relatives at similarity coefficient of0.58. Subsequently, AFLP marker results confirmedthat among evaluated close relatives of mangosteenG. malaccensis and G. porrecta consistently closer tomangosteen accessions clustering group compare toother close relatives.

DiscussionsSince AFLP markers produced higher polymorphic

characters (220 bands) compare to those of resultedby isozyme marker (22 polymorphic bands), AFLPmarker revealed higher genetic diversity 79%

compare to 62% that explained by isozyme marker.Cophenetic correlation value of both markers as highas 90% showed that the dendrogram generated fromboth markers have equal clustering patterndescended from the symqual matrix. The highestcophenetic correlation resulted by AFLP marker was0.978. This value showed correlation betweengrouping and similarity matrix was fit, and gave bestvalue to construct the grouping and arrangementsimilarity matrices (Table 3). However, groupingpattern in isozyme marker was slightly different tothose of AFLP marker, in terms of the number ofgroups, since isoenzymes generated four clusteringgroups compared to AFLP marker that generated sixclustering groups (Table 3).

The occurrence of genetic variability between andwithin individuals, within population and betweencultivars in cultivated species occurred by mutation,introgression, recombination, adaptation to newenvironment, and selection which occurs continually(Geleta et al., 2007). Genetic diversity withincultivated and wild plants is important to preventsome problems associated with cultivation failure.Cultivated plants can be improved by introduction ofwild relatives especially in the center of distribution,such as the mangosteen which is distributed inIndonesia and Malay Peninsula (Harlan and de Wet,1971; Hawkes, 1977).

High genetic diversity as represented bypolymorphic band percentage is not common formangosteen as an apomictic obligate, this might dueto several factors as accumulation of naturalmutation, repeated hybridization among mangosteenprogenitors Carman (2001), and ploidy developmentalprocesses. High variation among mangosteengenotype is a genetic potential to obtain high potentialgenotypes for specific purpose, which could be donethrough selection approach among superior trees inthe field (Sobir and Poerwanto, 2007).

Since G. malaccensis consistently showed closergenetic relationship with G. mangostana clusteringgroup in isozyme and AFLP markers, we conductedbands similarity proportion analysis that contributedby G. malaccensis, G. porrecta and G. hombronianawhich were estimated as mangosteen progenitoragainst the mangosteen based AFLP markers. G.malaccens is shared 53% similar band with G.

Table 3. Similarity coefficient value, cophenetic correlation, mangosteen group and close their relatives with isoenzyme andAFLP markers in similarity 58%.

Isoenzim AFLPSimilarity coefficient Value Group Accession Similarity coefficient Value Group Accession

Polymorphism (%) 88% I M, GM, GP Polymorphism (%) 100% I M, GPHighest value (%) 0.889 II GR Highest value (%) 0.773 II GM(Accessions) GM vs. L III GH & GC (Accessions) GP vs. L III MKLowest value (%) 0.2 IV GB Lowest value (%) 0.169 IV GH & GB(Accessions) GB vs. W (Accessions) GR vs. S V GCCophenetic correlation (r) 0.902 Cophenetic correlation (r) 0.978 VI GRNotes: M = mangosteen (G. mangostana), GM = G. malaccensis, L = Lampung mangosteen, GB = G. benthami, W=Wanayasa mangosteen, GP = G. porrecta, GR = G. rigida, GH = G. hombroniana, and S = Sukabumi mangosteen.

SOBIR et al. Isozymes and AFLP diversity of Indonesian mangosteen 167

G. porrecta shared 61.5 % similar band with G.mangostana, while G. hombroniana shared 50%similar band with G. mangostana. Moreover, if G.malaccensis and G. hombroniana simulated asprogenitor of G. mangostana, 33% of G mangostanabands could not explained by G. malaccensis and G.hombroniana, while if G. malaccensis and G. porrectasimulated as progenitor of G. mangostana, 29 % of G.mangostana bands could not explained by G.malaccensis and G. porrecta.

These result of above indicated that the proposalof G. malaccensis and G. hombroniana wereprogenitor of G. mangostana should be reviewedcarefully with more accurate evidences, since fruitmorphology of G. mangostana to fruit morphology ofG. porrecta, compare to those of G. hombroniana fruitcharacters (Sobir et al., 2009, unpublished data).

CONCLUSION

Isoenzyme assay employed four enzyme systemsand three primer combinations of AFLP marker on 13accessions of mangosteen and their close relativessuccessfully produced high polymorphism band andelucidate genetic variability at similarity coefficient of0.38 and 0.21 respectively. Both markers exhibitedsimilar clustering pattern, and grouping Gmangostana accessions in a clustering group. G.malaccensis and G. porrecta consistently in bothmarkers showed closer genetic relationship to G.mangostana clustering group compare to G.hombroniana that implies the assumption ofprogenitor of G. mangostana, should be reviewedwith more accurate data.

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Corresponding address:Jl. Gunung Salju Amban, Manokwari 98314, Papua BaratTel. +62-986-212095, Fax.: +62-.986-212095e-mail: [email protected]

Genetic Relationship of Sago Palm (Metroxylon sagu Rottb.) inIndonesia Based on RAPD Markers

BARAHIMA ABBAS1,, MUHAMMAD HASIM BINTORO2, SUDARSONO2, MEMEN SURAHMAN2, HIROSHI EHARA31Faculty of Agriculture and Technology, State University of Papua (UNIPA), Manokwari 98314, Indonesia

2 Faculty of Agriculture, Bogor Agricultural University (IPB), Bogor 16680, Indonesia3 Faculty of Bioresources, Mie University, 1577 Kurimamachiya,Tsu-city, Mie-Pref. 514-8507, Japan

Received: 8th March 2009. Accepted: 20th July 2009.

ABSTRACT

The areas of sago palm (Metroxylon sagu Rottb.) forest and cultivation in the world were estimated two million hectares andpredicted 50% of that areas located in Indonesia. Distribution of sago palm areas in Indonesia is not evenly distributed aswell as their diversities. Information of plant genetic diversities and genetic relationship is very important to be used forgermplasm collection and conservation. The objectives of research were revealed the genetic relationships of sago palm inIndonesia based on RAPD molecular markers. Fragments amplification PCR products were separated on 1.7% agarosegel, fixation in Ethidium Bromide, and visualized by using Densitograph. Genetic relationships of sago palm in Indonesiashowed that sample in individual level were inclined mixed among the other and just formed three groups. Geneticrelationship of sago palm population showed that samples populations from Jayapura, Serui, Sorong, Pontianak, and SelatPanjang were closely related each others based on phylogenetic analysis and formed clustered in one group, event thoughinclined to be formed two subgroups. Populations from Manokwari, Bogor, Ambon and Palopo were closed related eachothers, they were in one group. Genetic relationships in the level of island were showed sago palm from Papua,Kalimantan, and Sumatra closely related. Sago palms from Maluku were closed related with sago palm from Sulawesiwhereas sago palm from Java separated from the others. Based on this observation we proposed that Papua as centre ofsago palm diversities and the origin of sago palm in Indonesia. This research informed us the best way to decide sago palmplaces for germplasm of sago palm conservation activity.


Key words: genetic relationships, population, sago palm, RAPD, Indonesia.

INTRODUCTION

Indonesia has the biggest sago palm (Metroxylonsagu Rottb.) forest and cultivation as well as its rich ofgenetic diversities. The areas of sago palm forest andcultivation in the world were predicted two millionhectares and estimated 50% of that area located inIndonesia. Kertopermono (1996) reported that sagopalm areas in Indonesia were larger than proposed byFlach (1983). According to measurement ofKertopermono (1996), sago palm areas in Indonesiawere 1,528,917 ha and it was distributed into severallocations in Indonesia. The locations of sago palmareas in Indonesia were observed in the previousstudied, namely: Irian Jaya 1,406,469 ha, Ambon41,949 ha, Sulawesi 45,540 ha, Kalimantan 2,795 ha,West Java 292 ha, and Sumatra 31.872 ha. Thedistribution of sago palm areas in Indonesia was not

evenly distributed as well as their diversities. Flach(1983) predicted that sago palm diversities inIndonesia were found higher in Papua islands (NewGuinea) than other islands in Indonesia.

Information of plant genetic diversities is veryimportant to be used for germplasm collection andconservation. When germplasm conservation activityis done, information on genetic diversities areneeded, especially from the natural habitat to carriedout germplasm conservation efficiently. A popularDNA markers used for revealing genetic diversitiesand genetic relationships are Random AmplifiedPolymorphism DNA (RAPD) markers. The RAPDmarker is one of many techniques used for molecularbiology research. The advantages of RAPD markersare simpler in their preparation than other molecularmarkers. The other RAPD markers are easy appliedfor examining the diversities of organism (Powel etal., 1995; Colombo et al., 1998; Ferdinandez et al.,2001), because it is not using radioactive andrelatively chief (Powel et al., 1995).

Research which carried out for revealing geneticrelationships by using RAPD markers were reportedfor Sorghum bicolor L. (Agrama and Tuinstra, 2003),

ABBAS et al. Genetic relationship of sago palm in Indonesia 169

Brassica oleracea L (Graci et al., 2001), andMedicago sativa L. (Mengoni et al., 2000). Whereas,study for genetic structure of population was reportedfor Acacia raddiana Savi (Shrestha et al., 2002),Pimelodus spp. (Almeida et al., 2004), and Primulaelatior (L.) Oxlip (Jacquemyn et al., 2004).


Sago palm samples were collected from severalislands in Indonesia. A total 100 samples of sagopalm were collected from six islands and ninepopulations of sago palm centre in several islands inIndonesia. Location and geographical range of theselected sago palm stands were presented in Figure1. The populations and the numbers of samples thatwere used in this experiment were presented in Table1. Leaf samples were collected and preserved byusing silica gel granules in zip lock plastic accordingto previous reported procedures (Chase and Hill,

1991). Isolation and extraction of total DNA from driedsago palm leaf samples were conducted usingprocedures as described in Qiagen DNA extraction kit(Qiagen, 2003). The total DNA was stored in -20oC infreezer until ready for using.

PCR AmplificationRAPD primers used in this research were as

follows: P01 (GCG GCT GGA G), P02 (GTG ACGCCG C), P04 (CGT CTG CCC G), P06 (TTC CGCGGG C), P17 (ATG ACG ACG G), OPG02 (GGCATC GAG G), OPA04 (AAT CGG GCT G), OPAB04(GGC ACG CGT T), OPAA17 (GAG CCC GAC T),and OPAB18 (CTG GCG TGT C). PCR mixtures andcycles condition were followed procedures describedby Ehara et al. (2003) which has a little bitmodification such as 0.12 M, 0.63 U Ampli TaqGoldTM, 10 ng DNA genome, 1.7% agarose gels forseparating amplification fragments, and visualizationby using Densitograph, Bioinstrument ATTA.

Figure 1. The map of sampling sites of sago palm used (scale 1: 39,800,000). The cycles represent the populationsampling. A. Selat Panjang, B. Bogor, C. Pontianak, D. Palopo, E. Ambon, F. Sorong, G. Manokwari, H. Serui, I. Jayapura.

Table 1. The populations and the numbers of sample used

Island Population Numbers of samplePapua Jayapura 6, 7, 9, 11, 14, 24, 27, 34, 35, 49, 49, 50, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98,

99, 100Serui 1, 3, 5, 12, 18, 25, 26, 38, 43, 44, 47, 48, 73, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85Manokwari 2, 4, 9, 20, 21, and 22Sorong 8, 13, 17, 28, 69, 70, 71, 72, 74

Maluku Maluku 10, 41, 45Sulawesi Palopo 36, 37, 39, 40Kalimantan Pontianak 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68Jawa Bogor 15, 16Sumatra Selat Panjang 23, 29, 30, 31, 32, 33, 42

A

C

B

D EF

G

H I


Data analysisDissimilarity matrix was calculated by using distan-

ce coefficient. The dissimilarity matrix was employedto construct phylogenetic by the Unweighted Pair-Group Method Arithmetic Average (UPGMA), usingthe Sequential Agglomerative Hierarchical NestedCluster Analysis (SAHN-clustering, Sneath and Sokal,1973) and TREE program from NTSYS-pc, version2.02 packages (Rohlf, 1998). Bootstrap analysis withpermutation 10,000 times were performed by usingsoftware Tools for Genetic Analysis (TFPGA 1.3).Ordinate analysis calculated by usingMultidimensional Scaling (MDS) and performed byusing NTSYS 2.02 Package (Rohlf, 1998).

RESULTS AND DISCUSSIONS

RAPD PolymorphismPolymorphisms of RAPD amplification fragments

by using ten RAPD primers and performed in thePCR tools were resulted 86 numbers of polymorphicfragments and two to seven genotype numbers perpopulation. Samples DNA Fragments resulted byPCR were shown in Figure 2. High numbers of RAPDpolymorphisms and genotypes were found in thisobservation. These results were similarly with geneticdiversity of sago palm in the previous study, by Eharaet al. (2003) by using RAPD markers utilizing smallamount individual sago palm samples from Indonesiaand Malaysia. Fig 2 showed that the performancesamples of DNA bands were amplified by using 10primer sets. Numbers of fragment DNA band wereamplified from each primer, and it was ranging from 6to 12 polymorphic bands per primers and nomonomorphic DNA band was observed. Theaverages polymorphic DNA bands were calculated 9per primer. Primer P17 was resulted the highestnumbers of polymorphic DNA bands that was 12 DNAbands, whereas primers OPA04 and P06 producedthe lowest numbers of polymorphic DNA bands thatwere produced 6 polymorphic DNA bands perprimers. Base pairs sizes of DNA bands produce by10 primer sets were ranging from 150 bp (base pairs)to 1800 bp. Overall primers used in this observation

were suitable for studying genetic of sago palm. Theprevious of this observation applied more than 100RAPD primers sets.

Genetic relationships in the level of individualsGenetic relationships in individual levels showed thatthe samples divided into three groups based onphylogenetic construction (Figure 3) and threeclusters based on multidimensional scaling analysis(Figure 4). Numbers of individual samples associatedin group I were the sample number 2, 10, 13, 15, 16,17, 20, 21, 22, 23, 33, 34, 39, 40, 42, 43, 44, and 62;group II were the sample number 6, 9, 14, 24, 25, 26,27, 41, 49, 51, 58, 75, 95, and 97; group III were thesample number 1, 3, 4, 5, 7, 8, 11, 12, 18, 19, 28, 29,30, 31, 32, 35, 36, 37, 38, 45, 46, 47, 48, 50, 52, 53,54, 55, 56, 57, 59, 60, 61, 63, 64, 65, 66, 67, 68, 69,70, 71, 72, 73, 74, 76, 77, 78, 79, 80, 81, 82, 83, 84,85, 86, 87, 88, 89, 90, 91`, 92, 93, 94, 96, 98, 99, and100. The individual samples in group I and group IIIwere associated individual samples from overallpopulations. Group II individual samples associatedwith population from Jayapura, Serui, Manokwari,Ambon and Pontianak. These grouping were similarlywith sago palm grouping by Ehara et al. (2003) whichdivided sago palm samples from Indonesia andMalaysia into two groups and sub group based onRAPD markers. Papua islands in Indonesia wereshown that individual samples divided into threegroups also based on cp-DNA markers (Barahima etal., 2005). Based on our observation, we proposedthat sago palm in Indonesia classified into threegroups. Individuals grouping in the phylogeneticconstruction were based on genetic distances,grouping methods, and coefficient used orbootstrapping levels. In our observation showed thatthe different genetic markers used did not changegrouping pattern of sago palm. Some cases in themolecular analysis, the dissimilarities groupingpattern, by using the same markers or differentmarkers, were found frequently in the studied ofgenetic relationships (Ishikawa et al., 1992; Viard etal., 2001; Panda et al., 2003).

Figure 2. Performance of RAPD fragment by using OPAA17 primers on 1.7% agorose gels. Marker (M) and the number ofwell (10 to 64) indicated number of sago palm samples.

10 12 14 17 18 21 22 25 M 24 27 28 29 30 36 37 39 49 50 51 52 53 54 55 56 M 57 58 59 60 61 62 63 64


Figure 3. Phylogenetic of samples in the level of individuals based on 86 loci and 10 RAPD primers of 100 individualssamples by using UPGMA clusters and bootstrap by using 10,000 permutations.

Figure 4. Ordinate analysis of individual level by using MDS based on 86 loci, 10 RAPD primers, and 100 individuals ofsago palm. Two dimension scales (4A) and three dimensional scales (4B). Individual samples from Jayapura ( ), Serui ( ),Manokwari ( ), Sorong ( ), Ambon ( ), Palopo ( ), Potianak ( ), Bogor ( ), Selat Panjang ( ).

Genetic relationships in the level of populationsPhylogenetic construction show that sago palm

samples in the population levels was divided into twogroups, those were group I and II. The group I wasinclined to form two sub groups because bootstrapvalue was high (0.99) in one of finger phylogenetic(Figure 5) and two clusters based on MDS analysis(Figure 6). The group I included population samplefrom Jayapura, Serui, Sorong, Pontianak, and SelatPanjang. The group II was associated populationsample from Manokwari, Ambon, Palopo, and Bogor.The group I will be divided into two sub groups. Thesubgroup I included population from Jayapura, Serui,and Sorong and the subgroup II included populationfrom Pontianak and Selat Panjang. The geneticrelationships in the level of population showed thesame pattern with individual levels, even thoughsamples in the level of population just inclined to form

three groups, but solid pylogenetic construction onlyshowed two groups (Figure 4). Variation levels weredetected in this observation similarly with geneticvariation of Cynara scolymus L. by using RAPDmarkers (Lanteri et al. 2001) and Medicago sativa L.(Mengoni et al. 2000). The differences of relationshipsamong population probably were caused by outbreeding, so that populations become different.Population differences may be owing to pollenmigration (Latta and Mitton 1997). Generally,pollination of sago palm occurred a cross pollinationsince male and female flower mature in different oftime period (Jong, 1995). Cross pollination process insago palm may cause population different.

Association sample population from Jayapura,Serui, Sorong, Pontianak, and Selat Panjang to formone group in the phylogenetic construction probablyowing to sago palm interchange from one population

4A 4B

0.74

1.00

0.87

0.850.77

0.80

0.73

0.640.480.55

III

II

I

0.53

0.87


Figure 5. Phylogenetic of samples in the level of populations based on 86 loci and 10 RAPD primers of 100 individualssamples by using UPGMA clusters and bootstrap by using 10,000 permutations.

Figure 6. Ordinate analysis of population level by using MDS based on 86 loci, 10 RAPD primers, and 100 individuals ofsago palm. Two dimension scales dimension (6A) and three dimensional scales (6B). Populations from Jayapura ( ), Serui( ), Manokwari ( ), Sorong ( ), Ambon ( ), Palopo ( ), Potianak ( ), Bogor ( ), Selat Panjang ( ).

to another population which carried by people. In thisresearch we do not know exactly, when sago palmcame of exchange and where sago palm populationoriginated. Based on sago palm diversities andnatural stand we found that the largest variation andthe largest natural stand in the population fromPapua. Sago palm population from Jayapura wefound the largest variation and the largest vernacularname was given by local people. Matanubun et al.(2005) reported that there were 96 sago palmvarieties in Papua based on morphologycharacteristic and Yamamoto (2005) reported thatthere were 15 sago palm varieties in Jayapura basedon morphological characters. Population fromJayapura has the largest variation of sago palm.Based on that data, we can estimate that thepopulation origin of population in group I came fromJayapura population. Populations formed in group II,we predicted also caused by interchange individual ofsago palm in the past through people mobilizationfrom one place to another place. Therefore, thepopulation in one group such as group II haveaverage genetic distance closed each others. We

have no sufficient data to estimate the populationorigin in group II. This research gives us informationfor the best way to chose sago palm places forgermplasm of sago palm conservation activities.

Genetic relationships in the level of islandsThe genetic relationships of sago palm in the level

of island showed that it also formed three groups asshown on individual levels. Sago palm sample fromPapua, Kalimantan and Sumatra were observed andshow genetic distance closed each others, andformed Group I. Sample from Ambon and Sulawesiformed Group II, and sample from Java formed GroupIII in the phylogenetic construction. The geneticrelationships based on phylogenetic construction(Figure 7) and MDS analysis (Figure 8) showed thatsamples in island levels were closely related betweensamples from Papua, Kalimantan and Sumatra.Samples from Sulawesi islands were closely relatedwith samples from Ambon. Samples from Java islandwere separated with samples from the others islandbased on RAPD markers. There was very interestingphenomenon, at which we should pay attention,

Jayapura

Serui

Sorong

Pontianak

ManokwariBogor

AmbonPalopo

0.940.99

0.620.96

0.400.37

1.00

Group II

Selat Panjang

Group I

Subgroup I

Subgroup II

0.60

6A 6B


samples in the island levels formed the same groupwith samples from other islands, which havedistances far away each other. Those shown byPapua island were in the same group with Sumatraisland (Figure 7) at group I. This phenomenon may beoccurred owing to samples in individual levels fromPapua have genetic distances more closely thanindividual samples from Sumatra, which made totalgenetic distance between Papua and Sumatra closedeach others. If we estimated through migrationaspects, probably individual of sago palm from Papuamixed with sago palm individual from Sumatra in thepast by people mobilization/migration. During theDutch colonization in Indonesia, people alreadymoved from Sumatra to Papua or the other wayaround, with probably people carrying sago palmplant and growing at new places for anticipating foodcrisis in the future. Features of sago palm in Papuahave highest variation, largest sago palm forest,many wild types, and semi cultivated. Sago palmfeatures in another island in Indonesia Such asSumatra, Kalimatan, Java, Sulawesi, and Malukuwere found sago palm cultivated, semi cultivated, low

variation, no wild types, and no sago palm forest.Therefore we estimated the origin of sago palm inIndonesia come from Papua. The genetic distances ofsago palm from Papua were assayed closed withsago palm from Sumatra. Probably, sago palm fromPapua moved to Sumatra which carried out by peoplewhen they moved from Papua to Sumatra in the pastand formed a new population in the new places. Thisprediction may occur because RAPD markers whichused did not show as conservative as cpDNAmarkers which uniparental inherited (Ishikawa et al.,1992; Savolainen et al., 1995). RAPD markers aremolecular nuclear genome which related with DNArecombinant process and biparentally inherited (Viardet al., 2001). Therefore, RAPD markers are molecularmarkers which it have no longer conservative periodstime rather than cpDNA markers. In the previousstudies at different plants showed that higher variationwere found by using nuclear genome markers(RAPD, AFLP, ISSR, and nuclear SSR), then usingchloroplast genome markers such as cpDNA markers(Hultquist, 1996; Viard et al., 2001; Cronn et al., 2002;Panda et al., 2003).

Figure 7. Phylogenetic of samples in the level of islands based on 86 loci and 10 RAPD primers of 100 individuals samplesby using UPGMA clusters and bootstrap by using 10,000 permutations.

Figure 8. Ordinate analysis of island level by using MDS based on 86 loci, 10 RAPD primers, and 100 individuals of sagopalm. Two dimension scales dimension (8A) and three dimensional scales (8B). Samples from Papua ( ), Ambon ( ),Sulawesi ( ), Kalimantan ( ), Jawa ( ),Selat Panjang ( ).

Papua

Kalimantan

Sumatra

Ambon

Sulawesi

JawaGroup III

0.54

0.99

0.41

0.89

1.00

Group I

Group II

8A 8B


CONCLUSIONS

Genetic relationships of sago palm in Indonesiashowed that sago palm in individual level wereinclined to mix among the others, and just formedthree groups. Sago palm population from Jayapura,Serui, and Sorong were closely related; sago palmfrom Manokwari, Bogor, Ambon, and Palopo wereclosely related; and sago palm from Pontianak wasclosely related with sago palm from Selat Panjang. Inthe level of Islands which has long geographicaldistance showed that sago palm from Papua islandclosed related with sago palm from Kalimantan andSumatra island. Sago palm from Ambon closelyrelated with sago palm from Sulawesi, and sago palmfrom Jawa island not formed cluster with sago palmfrom the other islands. Thus, we proposed that Papuais as centre of sago palm diversity, and the origin ofsago palm in Indonesia. This research informed usthe best way to decide sago palm places, forgermplasm and sago palm conservation activity.

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Corresponding address:Kampus UNCEN WAENA, Jl. Kamp Wolker, Jayapura 99358Tel./Fax.: +62-967-572115email: [email protected].

Mycorrhizal Association of Terrestrial Orchids of CycloopsNature Reserve, Jayapura

VERENA AGUSTINI, SUPENI SUFAATI, SUHARNOBiologi Department, Faculty of Mathematics and Natural Science, Cenderawasih University (UNCEN), Jayapura 99358, Papua

Received: 14th December 2008. Accepted: 15th June 2009.

ABSTRACT

Study on exploration of mycorrhizal association of terrestrial orchid of Cycloops Nature Reserve, Jayapura was done. Theaims of this study were to collect terrestrial orchid and to isolate orchid mycorrhiza associated with it. Survey method wasused in this study. Isolation of orchid mycorrhiza was based on modified methods of Masuhara and Katsuya (1989). Theresult showed that there were 10 species of terrestrial orchid in this area. Eleven orchid mycorrhizal fungi were isolated fromfive terrestrial orchids. Among them, 6 isolates were associated with Geodorum sp. From the seventeen mycorrhizal fungi, 3isolates were identified, namely Rhizoctonia sp., Tulasnella sp., and Ceratorhiza sp, while the last fourteen isolates havenot been identified yet. Mostly, each isolate has a specific orchid host, except species G (sp. G) which associated withPhaius sp. and Plocoglottis sp.


Key words: terrestrial orchid, orchid mycorrhiza, Mt. Cycloops Nature Reserve, Jayapura.

INTRODUCTION

Papua contains very high level of plant diversity. Itmay have at least 20.000-25.000 species of vascularplants including orchids. 3000 species of orchids arefound in Papua, mostly are epiphytic. The explorationof orchids in Papua remains uncompleted due to acomplicated geographic mosaic. Some orchid specieshave restricted ranges as a consequence of thecomplex geologic history of the island and itsnumerous barriers to dispersals. One area whichshould be explored is Cycloops Nature Reserve(Cagar Alam Pegunungan Cycloops, CAPC). It istotally 22.520 ha (SK Mentan No.05/KPTS/UM/1978).

All orchids, epiphytic or terrestrial and autotrophicor heterotrophic heavily dependent on fungi for theirexistence. It is different with the symbiosis betweenplant and fungi called AM (arbuscular mycorrhizal),and also ECM (ectomycorrhizal). The role of the fungiis unique, which known to serve as orchidsmycorrhiza. Terrestrial orchids, in their habitatsrequire the presence of suitable fungi in the livingcells of the plant embryo and development ofmulticellular absorptive structures in order to developand mature successfully (Currah et al., 1990)

Many researches have been done for mycorrhizalof terrestrial orchids in temperate area, but only a fewin tropical area. Mycorrhizal fungi are associated withroot systems of more than 90% of terrestrial plantspecies in a mutual symbiosis. In nature, all orchidsutilize endomycorrhizal fungi to initiate seedgermination and seedling development. Theavailability of each fungi, therefore, is an absoluterequirement of orchid life cycle The orchids-fungussymbiosis is initiated when orchid seeds are infectedby a suitable fungus (Arditti, 1982; Rasmussen, 1995)

Orchids inoculated by fungi isolated from otherplant did not show any positive effect on seedlingdevelopment (Agustini and Kirenius, 2002).Dendrobium seedling inoculated with orchid-mycorrhiza isolated from other orchids shown a bettergrowth than non inoculated one (Agustini, 2003).Most of orchid-mycorrhiza is endomycorrhiza. Fungiassociate with photosynthetic orchid mostly belong tosubdivision Basidiomycotinae, class Hymenomycetes,genus Rhizoctonia. Otero et al. (2002) reported thatamong 9 species of Puerto Rican orchids there were108 Rhizoctonia like fungi which belong to Tulasnella,Ceratobasidium, and Thanatephorus.

Studies by Taylor and Bruns (1997) and Taylor etal. (2004) shown that 17 to 22 fungi species ofRussulaceae are associated with Corallorhizamaculata. Limodorum abortivum, an orchid grown inMediterranean is also associated with fungi belong tofamily Russulaceae (Girlanda et al., 2006).Russulaceae is basidiomycetes-ectomycorrhizal.


Orchids of CAPC still remain as one of the leaststudied (Pokja Cycloops, 2003), therefore some areassuch as surrounding Jayapura city, 6 species ofterrestrial-orchids were found (Numberi, 2005). Studyfor the rest of CAPC areas is needed. Because ofvirtuallyl lack of knowledge in the biodiversity of themycorrhizal fungi of tropical Orchidaceae, thedistribution and identification of fungi from variety ofterrestrial orchids were examined. The objectiveswere to make an inventory of terrestrial-orchids ofCPAC area, and to isolate and identify the naturallyoccurring mycorrhizal fungi of terrestrial orchids fromvarious habitats.


The status of orchids-mycorrhizaSurvey on status of orchids-mycorrhiza was done

due to determine whether any infection of fungi in theliving cells of the roots of the developing orchids ornot. Healthy roots of terrestrial-orchids were collectedfrom CPAC areas, namely: (i) UNCEN Campus atWaena (Kamp Walker and Buper), (ii) Sentani (Kemiriand Kampung Harapan), and (iii) downtown city ofJayapura. They were taken, wrapped in tissue paper,put in plastics bag and bring to the laboratory.

Collection and identification of terrestrial orchidsSample of terrestrial orchids were collected from

various locations of CPAC areas. They were collectedex situ on sterile media due to collect fungi from theorchids. The samples were identified at BotanyLaboratory, Cenderawasih University, Jayapura.Some references like Becker and Bakhuizen van denBrink (1963, 1965, 1968); Comber (1990); Segerback(1992); Schuiteman (1995); Mahyar and Sadili (2003);Banks (2004) were used.

Fungal isolationThe root segment taken from root tip was washed

with distilled water to remove any soil. For surfacesterilization they were treated with ethanol 70% for 30second. The roots were then cut into transversesection about 300 m thick and observed for thepresence of hyphal coil (polotons) on a glass slideunder a stereo-microscope in sterile condition. Thenthey were culture onto potato dextrose agar (PDA)media in petridishes, three pieces in each petridish.(Irawati, 2006; pers. comm). Mycorrhizal fungi wereisolated using a modification of Masuhara andKatsuya Methods (Manoch and Lohsomboon, 1991).After 1-2 days incubation, the hyphae will grow. If itshows any differences in shape and pattern ofgrowth, then they will be isolated and culture on PDAin order to separate the variety of the fungi. Purecultures were maintained on PDA slant.

Fungal identificationMacroscopic features examined were colony

growth pattern, color, and mycelia formation. Fungal

growth rate was measured from the colony on PDA.For microscopic examination, fertile hyphae weremounted in sterile water on a microscopic slide,covered with a cover slip, and examine under lightmicroscope. Hyphal and monilioid cells weremeasured. Literatures used in the fungal identificationwere Sharma et al. (2003) and Athipunyakom et al.(2004).


Terrestrial orchid speciesTen terrestrial orchids were found in this study.

Among the tenth species, 3 are found in campusareas, seven others are found in Sentani areas (Table1).

Table 1. The location of terrestrial orchids of CPAC

No. ofcollection Name of species Location

WAE-01WAE-02SEN-01SEN-02

Spathoglottis plicata L. CampusCampus (Buper)Sentani (Kemiri)Sentani (Harapan)

WAE-03WAE-04

Phaius tankervilleae CampusCampus

WAE-05SEN-10

Geodorum sp. CampusSentani (Harapan)

SEN-04 Calanthe sp.1 Sentani (Harapan)SEN-05 Calanthe sp.2 Sentani (Harapan)SEN-06 Plocoglottis sp.1 Sentani (Harapan)SEN-07 Plocoglottis sp.2 Sentani (Harapan)SEN-08 Plocoglottis sp.3 Sentani (Harapan)SEN-09 Paphiopedilum violascens Schltr. Sentani (Harapan)JAP-06 Macodes petola Jayapura

The result is slightly different with Numberisstudied on 2005. She found 6 species of terrestrialorchids growth at city of Jayapura areas includingcampus surrounding areas. 4 species namelySpathoglottis sp., Phaius sp., Geodorum sp., andMacodes sp. are found in Jayapura surroundingareas.

Two of them Spathoglottis sp. and Geodorum sp.were found in broader areas of study. Spathoglottissp. can be found in many habitats; from open areas toshading areas in forest either secondary or tertiary.The variety of habitat of Spathoglottis sp. make theflowers have different colors which were identified byNumberi (2005) as different species. It is stated insome literatures that wide ranges of habitat mightcaused the variety of color of flower.

Among the tenth species, some mycorrhizal fungihas been isolated just from 8 species, fungi from the2 species remains unisolated. The results of thepresent study showed that one orchid was associatedwith a number of mycorrhizal fungi, for exampleGeodorum densiflorum associated with 6 mycorrhizalfungi; Plocoglottis sp.3 associated with 3 mycorrhizal

AGUSTINI et al. Mycorrhiza of Cycloops terrestrial orchids 177

Table 2. Mycorrhizal fungi isolated from terrestrial orchids of CPAC.

No. ofcollection Species

Mycorrhizalfungi Habitat Location Isolate characteristics

WAE-01 Spathoglottis sp. Rhizoctoniasp.

Open areas Campus On PDA, colony growth slow, 4,5 cm in diameter after 4days incubation, thin mycelia, hyalin, globose, hyphaeseptate.

Tulasnellasp.

Open areas Campus On PDA colony growth slowly but faster thanRhizoctonia sp., mycelium white dense, hyphaebranches. Diameter 5,5 cm after 4 days incubation.Hyphae septate, rare, some of hyphae have branches,upright angles thicken. Conidia globe to ellipsoidal.

WAE-05 Geodorumdensiflorum

Sp.A *) Shading/canopy areas

Campus On PDA colony growth rapidly, mycelia dense, hyphaebranches rarely, end of hyphae bearing abundantconidia, globe.

Sp.B *) Shading/canopy areas

Campus On PDA colony growth slowly, thin mycelia, hyphaebranching often at nearly upright angles but morecommonly at 40-60o

Ceratorhizasp.

Shading/canopy areas

Campus On PDA colony growth slowly, mycelia thin, hyphaebranching, end of hyphae bearing abundant monilloidcells with typical mode of attachment in a chain 5-6globes.

Sp.D *) Shading/canopy areas

Campus On PDA colony growth more slowly, dense, circlegrowth, black-white. Mycelia thin, septate, abundantglobulus oil. Fungi isolated from terrestrial orchid

Sp.E *) Shading/canopy areas

Campus On PDA colony growth slow, circle growth, black andwhite. Concentric zonation, mycelia dense, septatedense, a few number of globulus oil.

Sp.F *) Shading/canopy areas

Campus On PDA colony growth slowly. Mycelia hyaline,branched, single globulus. A few number of coil.

WAE-03 Phaius sp. Sp.G *) Shading areas,next to creek.

Campus On PDA colony growth rapidly. Hyphae dense,branches, peloton. Hyphae septate, multinucleate, afew number of globulus.

SEN-04 Calanthe sp.1 Sp.H *) Secondaryforest

Harapan,Sentani

On PDA colony growth rapidly, densely hyphae,branches. Characteristics globulus with stem on rightand left of main hyphae.

SEN-05 Calanthe sp.2 Sp.J *) Secondaryforest

Harapan,Sentani

On PDA colony growth rapidly, white until optimalgrowth, turn on black color. Diameter 1 cm after 1 dayincubation, and 5.5 cm after 5 days of incubation.Hyphae dense, globulus branches.

SEN-06 Plocoglottis sp.1 Sp.I *) Secondaryforest

Harapan,Sentani

On PDA colony growth rapidly, dense, parallel growthhyphae, on tip of hyphae abundant granule.

SEN-07 Plocoglottis sp.2 unisolated Secondaryforest

Harapan,Sentani

-

SEN-08 Plocoglottis sp.3 Sp.G *) Secondaryforest

Harapan,Sentani

On PDA colony growth rapidly. Hyphae dense,branches, peloton. Hyphae septate, multinucleate, afew number of globulus.

Sp.K *) Secondaryforest

Harapan,Sentani

On PDA colony growth rapidly, white, dense at center,hyphae thin, branching upright, smaller branchesloosely arrange. Bearing abundant globulus, especiallyat the tip of growing hyphae.

Sp.L *) Secondaryforest

Harapan,Sentani

On PDA colony growth slowly after 15 days ofincubation the color turn in black, concentric zonation,dense and thin alternately. Mycelia growth fast anddense. Abundant granular.

SEN-09 Phapiopedilumviolascens Schltr.

Sp.M *) Secondaryforest

Harapan,Sentani

On PDA colony growth rapidly, white, with dense aerialmycelium in the centre. Hyphae septate, dense,granular rare. Host: Paphiopedilum violascens Schltr.

Sp.N *) Secondaryforest

Harapan,Sentani

On PDA colony growth rapidly, white, hyphae dense,branching densely, with abundant globular. Host:Paphiopedilum violascens Schltr.

JAP-06 Macodes sp. unisolated Secondaryforest

Jayapura -

Note: *) unidentified.


Figure 1. Six isolates taken from the root of terrestrial orchid Geodorum sp.: A. Sp.A, B. Sp.B, C. Ceratorhiza sp., D. Sp.D,E. Sp.E, and F. Sp.F (400x).

Figure 2. Reproductive portion of mycorrhiza. A. Hyphae of Rhizoctonia sp. 4 days on PDA. 400 x. B. Hyphae Sp.G taken fromPhaius sp., septate, multinucleate, globulus oil, 1000x. C. Conidia of Tulasnella sp., 400 x. D. Hyphae supporting conidiospores, 100x.

Figure 3. Colony of mycorrhiza. A. Sp.I: colony on PDA showing concentric zonation, 100x; B. Sp.I: abundant granule on tipof hyphae, 100x; C. Sp.J: colony on PDA; D. Sp.J: branches hyphae with globulus, 400x; E. Sp.L: colony on PDA showingconcentric zonation and black in color after 15 days incubation; F. Sp.L: hyphae with abundant granule, 100x; G. Sp.M:dense and septate hyphae with few granule, 1000x; H. Sp.N: dense and branching hyphae with abundant granule, 100x.

A C D

HE

B

F G

D

BC D

F

A B C

A C D E

AGUSTINI et al. Mycorrhiza of Cycloops terrestrial orchids 179

fungi (sp.G, sp.K, and sp.L); Spathoglottis andPaphiopedilum violascens associated with 2mycorrhizal fungi each. The study also found that anumber of mycorrhizal fungi were associated with asingle orchid host, for instance Rhizoctonia sp. andTulasnella sp. are associated with Spathoglottiswhereas Mycorrhizal fungi sp.M and sp.N areassociated with Paphiopedilum violascens (Figure3).The other orchids are associated with singlefungus. This study indicated that mycorrhiza sp.Gwas isolated from Phaius sp. and also fromPlocoglottis sp.3.

This study showed that from the two orchidsPlocoglottis sp.2 and Macodes sp., mycorrhizal fungiremained unisolated. There are 17 mycorrhizal fungiisolated from 8 orchids of CPAC areas (Table 2).Athipunyakom et al. (2004) did a similar study, andfound 14 mycorrhizal fungi from eleven terrestrialorchids in Thailand, among them are Rhizoctoniaglobularis, Ceratorhiza sp., and Tulasnella sp.

Association of mycorrhiza and terrestrial orchidThe results of the study shown that a number of

mycorrhizal fungi were associated with only oneorchid. There were six mycorrhizal fungi associatedwith Geodorum sp. (Figure 1). Rhizoctonia sp. andTulasnella sp. are symbiont of Spathoglottis.(Figure2). According to Athipunyakom et al. (2004),Tulasnella sp. Is also associated with Cymbidiumtracyanum, in root of Calanthe sp. was found fungiEpulorhiza and Ceratorhiza, while in this studyCeratorhiza fungi were isolated from Geodorum sp.Fungi sp.G are associated with both Phaius andPlocoglottis sp.3. Kristiansen et al. (2004) also founda similar situation that many fungi were associatedwith one specific orchid.

Almost all mycorrhiza associated with terrestrialorchid is Rhizoctonia including anamorphic ofTulasnella, Ceratobasidium, and Thanatephorus(Otero et al., 2002; Bonnardeaux et al., 2007). Fungiknown to be associated with Spathoglottis sp. isRhizoctonia, similar to study done by Tan et al. (1998)using terrestrial orchid Spathoglottis plicata. Heinoculated Rhizoctonia AM9 on the media togerminate seed. The study showed that there isimportant role of the fungi on Spathoglottis plicataseed germination. Hayakawa et al. (1999) showeddifferent results, there was no significant effect of theisolate on in vitro seed germination.

In contrast, this study indicated that one fungimerely associated with a single orchid host. Calanthesp.1 and Sp.H and Calanthe sp.2 and Sp.J whichboth remained unidentified (Table 2). Study ofsymbiosis of fungi and orchid is extremely unique,mainly Pterostylis, Caladenia and Thelymitra. Thesefungi grow in very close habitat but they have differenthost, namely Ceratobasidium, Sebacina andTulasnella (Andersen and Rasmussen, 1996). TheFungi Rhizoctonia is known as a symbiont ofSpathoglottis sp, and is found in Pinus radiata root as

well. Furthermore, Bidartondo et al. (2004), reportedthat orchids grow under canopy can be associatedboth with fungi mycorrhiza and trees at thesurrounding habitat. Fungi which are associated withorchid are basidiomycetes. Some of them aresaprophytic, ectomycorrhiza, and plant parasitic(Rasmussen, 2004), while Yamato et al. (2005) saidthat achroroplilous Epipogium roseum also knownassociated with Coprinus which is saprophytic fungi.

The specific relationship between fungi and orchidalso reported by Rasmussen (2004). The specificitywas found from taxon species to subtribe (Warcup,1981). Photosynthetic orchids showed a highspecificity in association of mycorrhizal-orchids(Shefferson et al., 2005). Specificity possibly leads tohigh rates of orchid seed germination and a moreefficient physiological association when theinteraction is fully functional (Bonnardeaux et al.,2007). Mycoheterotrophic orchid Corallorhizamaculata was associated with twenty two fungibelong to family Russulaceae which isectomycorrhizal in plants (Taylor et al., 2004).

In this study, transversal root segments ofterrestrial orchids were used as a source ofmycorrhizal fungi, this method were also used byShan et al. (2002) and Bonnardeaux et al. (2007).Other isolates source of orchids-fungi were hyphalcoils (peloton) taken from longitudinal sections ofroots (Athipunyakom et al., 2004; Bonnardeaux et al.,2007).

CONCLUSION

Ten terrestrial orchids were found in Cagar AlamPegunungan Cycloops, Jayapura. From eight of theten orchids there were seventeen mycorrhizalorchids, three of them are identified as Rhizoctoniasp., Tulasnella sp., and Ceratorhiza sp. Dari Amongthem six fungi isolated from a single orchid Geodorumsp. In this study the specificity was found in mostmycorrhizal-fungi except Sp.G which found in twodifferent terrestrial orchid species namely Phaius sp.and Plocoglottis sp.

ACKNOWLEDGEMENTS

We thank to the Directorate General of HigherEducation, Ministry of National Education throughFundamental Research Grant, 2007 and IndonesiaManaging Higher Education for Relevance andEfficiency Project (I-MHERE Project) Sub-ComponentB.1. Batch II, through Research Grant Program 2007for financial support, and we also would like to thankto Ira Aldila Putri and Dewi Katemba for theirlaboratory work.


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Alamat korespondensi:Jl. Syech Abdul Rauf, Darussalam, Banda Aceh 23111Tel. +62-0651-7410248, Fax. +62-0651-7551381e-mail: [email protected]

Growth Rate of Acropora formosa Fragments that Transplantedon Artificial Substrate Made from Coral Rubble

NUR FADLIMarine Science Department, Faculty of Science, Syiah Kuala University, Banda Aceh 23111, Indonesia

Received: 25th March 2009. Accepted: 16th June 2009.

ABSTRACT

Coral reefs play an important physiological and ecological role in coastal ecosystems such as providing natural breakwatersthat protect shorelines and human settlements from waves and storm. Corals killed by tsunami, waves and storms are oftendegraded into rubble. This rubble is dynamic, easily shifted by currents and storms, which effectively forms killing fields forcoral juveniles, hindering coral recovery. In order to rehabilitate coral reefs, artificial substrates are used both for coraltransplantation and recruitment. Unfortunately, most artificial substrates are expensive and use land-based material such asconcrete/cement-bases. In order to develop a new low-cost artificial substrate that can replace concrete/cement-base as amedia for coral transplantation, modified coral rubble was tested in a pilot study in Seribu Island, Jakarta. Two different nets(nylon and polyethylene) were used to form rubble into a compact shape, stable and strong substrate. The stability of therubble and the complexity of the surface which is created by the net make this substrate suitable for coral transplantation.Additionally, from an economic perspective the nets are very cheap and locally available. In a number of experiments,modified coral rubble successfully replaced the concrete/cement-base as a media for coral transplantation. The coraltransplants were growing over time. With this method, we can try to rehabilitate the degraded coral reef destroyed bytsunami or other factors with material that already is available at the site and with less money. However, this approachrequires testing at additional sites and for longer periods, to determine the replicability of the results.


Key words: cement base, coral, rubble, transplantation.

INTRODUCTION

Coral reefs are regarded as one of the mostdiverse, complex (Buddemeier et al., 2004; Veron,1995) and productive ecosystems (Burke et al., 2002;Tomascik et al., 1997) on earth. At least, 794 speciesof scleractinian corals are known to build coral reefs(Spalding et al., 2001). The majority of coral reefs arelocated in tropical and subtropical regions between22. 5N and 22. 5S latitude, with the center ofmaximum coral diversity in the Southeast Asianregion (Buddemeier et al., 2004). The Indonesianarchipelago with more than 17,500 islands and acoastline in excess of 80,000 km, is one of the largestcountries with coral reefs in the region (Burke et al.,2002; Nontji, 2004; Tomascik et al., 1997).Approximately 18% of the worlds coral reefs arelocated in Indonesia. More than 480 species of hardcorals (which represents 60% of the described coralin the world) are located in Indonesia (Burke et al.,2002). Unfortunately, in 2003, it was estimated that

only 7% of Indonesias reefs remained in an excellentstate. Over 27% were in fair condition, and more than36% were reported to be in poor condition (Nontji,2004).

There are many factors involved in degradation ofcoral reef ecosystem in Indonesia. Some of them arenatural, but several factors are anthropogenic. One ofthe common threats is blast fishing. In remote areas,were law enforcement is minimal, blast fishing is morecommonly practiced (Erdmann, 1998; Kunzmann,2002). Blast fishing has serious negative impacts oncorals because the blast shatters the coral skeleton,which leads to mass fragmentation. Some of thefragments may initially survive for several months buteventually die (Fox et al., 2003). A typical 1 kg beerbottle bomb can create a rubble field of 1-2 m indiameter (Burke et al., 2002). Furthermore, blastfishing may leave fields of rubble that shift in thecurrent, abrading or burying new coral recruits, andthereby slow down or prevent the reef from recoveryeven when an area is protected from further blasting(Fox et al., 2005). In the Philippines, many rubblefields virtually show no hard coral cover upon 20-30years post-blasting (Raymundo et al., 2007). Due tothis reason, artificial substrates are always used incoral transplantation in areas of unconsolidated


sediment and water movement. Unfortunately, mostrehabilitation techniques are expensive and laborintensive (Clark and Edwards, 1995; Edwards andClark, 1998). Researchers comparing various coralrestoration methods found that costs could rangefrom US$13,000 to more than US$100 million/ha(Spurgeon and Lindahl, 2000). Unsurprisingly, themethods are not suitable for developing countries likeIndonesia (Fox et al., 2005).

Artificial substrates should possess the ability toavoid the abrasion, dislodgement and transport due towater movement (Lindahl, 2003), and be placed highenough above the bottom substrate to minimize burialand abrasion (Fox et al., 2005). As long as theartificial substrate can accommodate these problems,different material can be used. In order to develop anew low-cost method of artificial substrate as a mediafor coral transplantation and coral recruitment a net isused to modify coral rubble into suitable media fortransplantation. The net will tie and make the rubble acompact shape, stable and strong substrate. Thestability of the rubble and the complexity of thesurface shape, which are created by the net mayincrease the natural coral recruitment (Edwards andClark, 1998; Raymundo et al., 2007). Furthermore,coral rubble provides an appropriate biofilm for larvaeto settle (Harrington et al., 2004; Mundy, 2000).Additionally, from an economic perspective the netsare very cheap and locally available.


The main study was carried out from the first weekof September 2007 until the second week of January2008. The experiment was located on the reef-flat onthe western side of Panggang Island (located in themid region of Seribu Island); about 300 m from theisland (Figure 1). The western part of PanggangIsland is a sandy plateau, which is part of the reef-flatzone. The reef-flat, which is composed of limestone,is covered with coral rubble and sand. The coral inthis site is considered in poor condition (coral cover 25%) 4 x 20 m2 line intercept transect in each depth,(English et al., 1997), with dead coral cover reachingalmost 50% in both (6 and 10 m) depths. In addition,the rubble covers 25% of the site. The immenseavailability of rubble in this site provides enoughmaterial for the experiment. Additionally, most of thecoral farmers place their coral farming in this area,indicating that this site is suitable for coraltransplantation.

For the experiment, three different artificialsubstrates with approximately equal size (size~30x50x10 cm3) were used: the cement base(cement-base), rubble which was tied together withnylon net (nylon + rubble) and rubble which was tiedtogether by polyethylene net (polyethylene + rubble)(Figure 2 and 3). The cement-based substrates wereconsidered as the control since this substrate was

commonly used as a medium for coral transplantationin Indonesian waters especially in the Seribu Island.In addition, the nets that were used in this experimentwere locally available with a 2 cm diameter meshsize. The rubble which was used to form the rubblesubstrates was taken from the water on the westernpart of the Panggang Island. The rubble was filled intothe net on the land and left for 2-3 days. The ironsticks (diameter = 10 mm) were used as a media toattach the coral fragment onto the substrate. Thesedifferent artificial substrates constituted three differenttreatments. Afterward, on each artificial substrate, fiveof coral fragments were interspersed at approximatelyequal intervals. The coral were tied directly to the ironsticks using plastic cable ties. The artificial substrateswere placed into two different depths (6 m and 10 m),representative of shallow and deep water. Anexception was the cement-base substrates, where 8substrates in 6 m and 10 substrates in 10 m weredeployed due to technical problems. The substrateswere deployed in the experimental site by using afisherman boat. The total sample size was 180fragments in 6 m and 190 fragments in 10 m. TheSCUBA gear was used to put the constructions inposition.

The species used during the experiment wasidentified as Acropora formosa. Fragments for theexperiments were collected from a donor site about100 m away from the experimental site. Donorcolonies were located at about 2-3 m depth. For everysurviving fragment, the change of the main branch inlinear extension (the total length from the apical tip tothe net) was measured with plastic Vernier calipers(0.01 mm error margin) for every sampling intervaland the overall duration.

For the calculation of growth rates per day, themean increment of all surviving fragments pertreatment per time was used, divided by the numberof days between two samplings. Fragments showingnegative growth between surveys were notconsidered in calculation of average growth rates tomake sure that only growth was considered. In orderto determine if the growth rates of the fragments weredifferent between substrate, the mean increment perday of transplants for each type of substrate wastested by Wilcoxon Rank Sum test. Each depth wasexamined separately. All data used in the statisticalanalysis were analyzed using JMP 7.0.1 software(trial version).


ResultsIn a number of experiments, the transplants were

increasing their length over time. Some fragmentswere also increasing the number of branchesindicating that they can grow on the substrates. Thestability of the rubble which is created by the net is

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