Biodiversitas vol. 10, no. 2, April 2009 by Biodiversitas Unsjournals

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BIODIVERSITAS Volume 10, Number 2, April 2009 Pages: 55-62

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100201

PCR-Based EST Mapping in Wheat (Triticum aestivum L.) 1,♥

1,2

TATIK CHIKMAWATI , MIFTAHUDIN , J. PERRY GUSTAFSON

Biology Department, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University (IPB), Bogor 16680, Indonesia Agronomy Department, University of Columbia, Columbia, MO 65211, USA, and USDA-ARS, Plant Genetics Research Unit, University of Missouri-Columbia, Columbia, MO 65201, USA

Received: 15th February 2008. Accepted: 28th March 2008.

ABSTRACT Mapping expressed sequence tags (ESTs) to hexaploid wheat is aimed to reveal the structure and function of the hexaploid wheat genome. Sixty eight ESTs representing 26 genes were mapped into all seven homologous chromosome groups of wheat (Triticum aestivum L) using a polymerase chain reaction technique. The majority of the ESTs were mapped to homologous chromosome group 2, and the least were mapped to homologous chromosome group 6. Comparative analysis between the EST map from this study and the EST map based on RFLPs showed 14 genes that have been mapped by both approaches were mapped to the same arm of the same homologous chromosome, which indicated that using PCRbased ESTs was a reliable approach in mapping ESTs in hexaploid wheat. © 2009 Biodiversitas, Journal of Biological Diversity Key words: wheat, EST, PCR, homologous.

INTRODUCTION Expressed sequence tag (EST), a single-pass sequence of cDNA, can be used to study structural and functional genomics. Expressed sequence tag databases are now becoming the basis for genomic approaches to drug discovery, plant and animal genetic improvement and the study of human genetic diseases (Messing and Llaca, 1998; Saier, 1998). Expressed sequence tag databases are also valuable sources for the construction of synthetic genome linkage maps of expressed genes among related species (Cato et al., 2001). Earlier methods for mapping ESTs involved RFLPs as hybridization probes. This approach has been augmented by more efficient PCR-based approaches where EST-specific primers are used to amplify coding sequences. A PCR-based approach is considered faster, cheaper, safer (no need radioisotopes), and need only a small amount of DNA as compared to RFLPs (Cato et al., 2001). The critical step in PCR-based EST mapping is the construction of specific primer combinations that produce a unique and polymorphic fragment among genotypes used in a study. The polymorphic band can be generated using restriction enzymes or single-stranded conformational polymorphisms (SSCPs) (Fischer and

 Corresponding address: IPB Campus at Darmaga, Bogor 16680 Tel. & Fax.: +62-251-8622833 e-mail: tchikmawati@yahoo.com

Lerman, 1983; Harry et al., 1998; Orita et al., 1989). The more difficult situation is when primers are designed from wheat (Triticum aestivum L.) ESTs in projects aimed to map hexaploid wheat. Most of the genes in hexaploid wheat are present on at least two of these homologous chromosomes. To locate ESTs to a specific chromosome, the primers should amplify a specific gene, produce a single band (if possible), and only amplify from one genome. The primers should also accommodate the possibility to locate the same gene to homologous chromosomes from other genomes. Today there are over 499,000 wheat ESTs that have been developed from different tissues from many different libraries, and available in wheat EST databases. Among those sequences, there are some EST sequences that are duplications and are derived from the same gene from the same chromosome. There are some EST sequences that show single nucleotide polymorphisms (SNPs) when they are aligned to each other, suggesting that those ESTs were derived from the same gene family from different homologous chromosomes or a different chromosome group. Sets of primer combinations could be generated to facilitate PCR-based EST mapping in the wheat genome by searching and employing the SNPs located to select ESTs. Hexaploid wheat is comprised of the three closely related genomes that facilitate a massive amount of gene buffering. This buffering allows hexaploid wheat to tolerate deletion of gene, gene complexes, chromosome arm segments, a chromosome arm, or the entire single chromosome for particular genome.

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Sears (1954) developed a series of ditelocentric lines, which are a series of wheat lines in which each line has a chromosome arm deletion for a particular chromosome. He also developed a series of nullisomic-tetrasomic lines, which is each line missing one entire chromosome for a particular genome, and has two extra chromosomes of one of its homologous chromosomes. These aneuploid stocks could be used to locate genes to a particular chromosome and chromosome arm. Physical mapping on wheat chromosome arm can be refined into a specific chromosome arm segment by using a series of wheat deletion stocks. Endo (1988) developed a series of wheat deletion stocks by introducing Aegilops chromosomes into a wheat background which caused deletions to be randomly established in wheat chromosome arms. There are over 436 deletion lines that have been isolated and used to chromosome bin map in the wheat genome. In this study, those aneuploid wheat stocks, including nullisomictetrasomic, ditelosomic, and deletion lines were used to physically map ESTs to wheat genomes. The objectives of this study were: (i) to develop primers from wheat ESTs that can be used to map ESTs using a PCR technique; (ii) to physically map wheat ESTs to the wheat genome using wheat deletion lines; (iii) to compare the PCR-based and RFLP-based mapping of ESTs to the wheat genome. This study was conducted in collaboration with Dr. D.J. Somers from Agriculture and Agri-Food Canada, Cereal Research Centre, Winnipeg, Canada.

MATERIALS AND METHODS Plant materials Plant materials included 45 aneuploid lines involving 21 ‘Chinese Spring’ wheat nullisomictetrasomic lines, and 24 Chinese Spring wheat ditelosomic lines from the USDA-Sears collection (University of Missouri-Columbia), and 101 Chinese Spring wheat deletion lines from Dr. B. Gill’s collection (Kansas State University, USA). DNA isolation DNA was extracted from young freeze-dried leaf tissue using methodology from Saghai-Maroof et al. (1994). Primer design All primers were designed in Dr. Somers's laboratory. Designing the EST primers required several steps. First, EST sequences were selected from wheat EST-National Science Foundation (NSF) and the International Triticeae EST Consortium (ITEC) databases. Second, the selected EST sequences were aligned with other ESTs to locate any SNPs. Only alignments that showed above 95% identity over 400 bases were used. Third, the primers were designed using the 20 nucleotides before a polymorphic nucleotide, and the last base on the 3’ end of each primer was the polymorphic nucleotide.

Each primer pair was given code number based on the order of the time made, called a blinded name. The last step involved checking the primers by using them to amplify Chinese Spring wheat genomic DNA to see whether the primers worked or not. The working primers were mapped using the Chinese Spring nullisomic stocks to assign the chromosome location, and then bin mapped using the Chinese Spring ditelosomic and deletion lines. The blinded names, primer names, sequences of the EST primer combinations, and the size of PCR product are listed in Table 1. Amplification of genomic DNA using EST primers Polymerase chain reaction amplifications were carried out in a volume of 20 uL (non-radioactive) in a Tetrad Thermocycler. The reaction mixture contained 24 ng of template DNA, 10 pmol of each primer, 0.8 nM of dNTPs, 1 U Taq DNA polymerase in 1x reaction of PCR buffer (10 mM Tris HCl, 50 mM KCl, 1.5 mM MgCl2, pH 8.3). The PCR process used a touchdown PCR program where the annealing o o o temperature is dropped 1 per cycle from 64 to 55 C o for first nine cycles then remains a constant 55 C for the remaining 21 cycles. Amplification products were mixed with 1:5 volume of loading buffer (50% glycerol, 0.5% bromophenol blue, 1 mM EDTA), and then the PCR products were resolved in a 1% agarose gel in 1xTAE buffer for 1 hour. Chromosome bin mapping of ESTs Figure 1 shows how EST 152 was mapped to a specific deletion bin on wheat chromosome 2D. Nullisomic-tetrasomic lines were used to locate which chromosome had the EST. For example, N2DT2A means this line has two sets of chromosome 2A instead of one set of chromosome 2A and one set of chromosome 2D. No PCR product resulted from the N2DT2A line, indicating EST 152 resided on chromosome 2D. The ditelosomic lines were used to locate on which arm the EST was located. We used ditelo (Dt) long arm (DL) and Dt short arm (DS). For example, Dt2DS means the line has only the short arm of chromosome 2D. The absence of PCR product in a Dt2DS line indicated EST 152 was located on the long arm of chromosome 2D. The deletion lines (DL) were used to find the exact bin location of the EST. Figure 1 shows that the EST 152 band was absent on deletion 2DL3 lines, but present on 2DS1, 2DS3, 2DS5, and 2DL9. This indicated that the exact location of EST 152 is in Bin 2DL3-0.49-0.76. Comparative map between PCR- and RFLP-based EST mapping The ESTs mapped using PCR-based technology were compared with the ESTs mapped using RFLPbased technology to establish the reliability of PCRbased technology for mapping ESTs on hexaploid genome. The wheat EST-NSF database (http://wheat.pw.usda.gov/cgi-bin/westsql) was used to search for the colinearity between two EST maps.

CHIKMAWATI et al. â&#x20AC;&#x201C; PCR-based EST mapping of Triticum aestivum

Table 1. The EST accession numbers, blinded names, primer names, primer sequences, and the size of PCRproducts. EST Blinded Primer Product Primer sequence accession name name size (bp) BE500218 11F F063 CTAGCAGCTTAAGGAACTTC 275 11R R271 GCAGGATCAGCACCGTGCCC BF203011 21F F174 GCACCGTCAGCACACGCAAA 200 21R R338 TCAGGAACACCACGCGCTTC BF202619 22F F073 TCGAAGAGGCGCTCGGCATA 325 22R R261 CACCGTGCCCGGTGTGATGA BF202379 31F F102 TCGACTCTGGCAAGTCGACA 250 31R R326 TCGACTCTGGCAAGTCGACA BF483165 32F F048 TCGACTCTGGCAAGTCGACC 300 32R R379 CAATGATGAGCACAGCACAA BE500396 42F F103 CGGCGAGGAAGTCGACCGGA 300 42R R334 CGGCCTCCTGCAGCGCGGAC BE500501 51F F064 CCTTCTTCAGCGAGACTGGG 175 51R R391 CACGGGCGAAGTTGTTGGCT BF483613 52F F076 AGACTGGGGCTGGGAAGCAC 300 52R R448 TCCTGATACGATCAAGGCAG BE500375 53F F278 ATGTCCCCCGTGCTGTCTTT 380 53R R546 GGCGCTCCAGGAGAAGCGAG BF482753 54F F087 GGAAGCATGTCCCCCGCGCG 325 54R R268 AGACCAGTGCAGTTGTCTGC BF482680 55F F247 AGATGCCCGGTGACAAGACT 425 55R R541 CAGTTCCACCTCCAACAGCA BF482845 56F F228 GATGGACAGATGCCCGGTGG 425 56R R520 TGAAGACGAGGAATCCCTGA BE637755 61F F071 AATCTTATGGATCAAGGTGT 550 61R R422 TGTGGATCTGGGTGGTGTCC BF482523 62F F093 GCCGCCCCAACCCCATGACC 400 62R R279 CAGCCTCATCGATTTGGATA BF483171 81F F061 AGTACCAGGTGGTGGGTCGT 1000 81R R322 TCTGCTCCACGGCACCATTT BE499999 82F F124 TGAAGCTCTGGGCCACCAAT 1000 82R R287 TCTGCTCCACGGCACCATTC BF484421 83F F091 CGCCCGGCGATGAGCAGCCG 1000 83R R287 TCTGCTCCACGGCACCATTC BF484421 84F F106 TCTGGGCGACCAACGAGGTG 1000 84R R242 ACATGTTGTGGTAGCCGGTT BE498885 91F F187 AATCGGAAAAGGCCCCCTAT 300 91R R243 TCATCGCTGCCCTCATCGTC BM140580 101F F034 TGGTCCTTATCCTTGCCATT 800 101R R526 TGTCCCAGGCCCTTCCACTA BM140371 102F F058 TGGTCCTTATCATTGCCATC 800 102R R550 TGTCCCAGGCCCTTCCACTC BF484255 103F F105 TCCCTTCATGGTCCTTATCA 800 103R R494 TTCCCTTGATCCTTGCGAAG BM137809 131F F142 CGTATGTGCTATCTGAGTCC 250 131R R295 CGCCAGACTTCAGGTGGCCG BF202081 132F F100 CTGAGGCATCAATCTTTGCT 375 132R R347 CACCAGACTTCAGGTGGCCA BF202081 133F F245 CCAACAAGGACTTCGACTCC 200 133R R211 GGAAGATGAACATCCCACGA BM134418 141F F059 TCGACAGGAGGCCAAAGGCG 500 141R R342 AGAGGTTGGTGTCCTCGAAG BM134418 142F F160 CGAGGAAGTCTGCGCCGACG 250 142R R367 TGTCCTCGAACAGCCCCACC BE499017 143F F174 AGTCGGCGCCGACCACCGGG 200 143R R318 CGTGGCTCTGGAAGCGGAGA BE500314 151F F023 GTGGCAATGCCTATGTGATT 250 151R R367 AGGGCTCGAAGTCGAAGTCG BM138609 152F F051 CTGTCTTCTTCAAGACTCAA 400 152R R384 TGCAGTCGTATGCCTCAGCT BM138609 153F F201 CTTCAAGACTCAAGCTGATA 250 153R R401 CGCCGTAGGCCAGGGCGGAG BE499318 161F F212 AGGTTTCTACCTCGAAGACA 400 161R R425 TGAGCTGGGTCAGGATAGCA BE423540 172F F097 AAGCTCGTTCTGTTCAGGAC 300

172R BQ240701 181F 181R BQ240701 182F 182R BF202965 201F 201R BE499290 211F 211R BQ620117 221F 221R BF202081 222F 222R BF202198 223F 223R BF482340 224F 224R BQ619858 231F 231R BG907231 251F 251R BG904361 253F 253R BG909863 261F 261R BF202040 271F 271R BF482437 272F 272R BQ620354 273F 273R BG906628 281F 281R BM140514 283F 283R BE422799 292F 292R BE499039 301F 301R BM138684 302F 302R BG904954 311F 311R BG906277 321F 321R BE424825 322F 322R BG907633 331F 331R BG910130 333F 333R BG906045 361F 361R BE424633 401F 401R BG904428 402F 402R BG904212 403F 403R BG905429 411F 411R BG905429 412F 412R BE425039 421F 421R BQ238323 422F 422R BQ620599 431F 431R BQ237808 432F 432R

R296 F129 R394 F129 R411 F151 R342 F125 R512 F039 R406 F099 R599 F056 R341 F056 R346 F180 R416 F088 R291 F132 R462 F074 R346 F083 R372 F062 R362 F141 R462 F086 R215 F067 R226 F264 R166 F027 R225 F096 R229 F187 R491 F274 R419 F234 R026 F205 R431 F552 R328 F338 R560 F044 R274 F014 R337 F170 R472 F139 R463 F135 R459 F209 R296 F275 R539 F080 R500 F477 R407

AGGACCTTGCACTCCTTGTC GCGTGAGGGAGCGCATCGGT 750 CCCTCACATACTCCCAGACG GCGTGAGGGAGCGCATCGGT 800 GCAAATACTCAGCGACGCTC CCGCCGCCTCGCCGGAGCGG 600 AGAGGCCCTTGACGATCCCT AGACCGGCTGCCAGTCTCGT 400 GGTAGTCAAACTTGCACTCA GGATGTTCATCTTCCCCGGT 400 GTCACATGGATGGTGGAGAA ATGTCCTGAACCGCTACTTT 300 AGGGCTCGAAGTCGAAGTCG GGATGTTCATCTTCCCCGGC 300 CCATCTCGGGAATGATGTCG GGATGTTCATCTTCCCCGGC 325 AGACCTCCATCTCAGGGATA ACCGCAGTTGCCTTGATATA 300 GCTTCGACCCACTGGGCTTG GGTACATACGCTGGAGAAAT 200 TCTTCTACTTCGAGGCCGGA ACACACCACCCCAAAGAAGA 350 ACTTCGCCAACTTCACCGGT CGCCGACGCAGCGAGCTCTT 400 CGACCTTCTCGGCGTAAAGT GCGCGGCCATCTCCATCTCA 550 CCTTAGCAGCAAGGCTGGTT CCCCTCTATCCGACTTCGGG 600 CCTTAGCAGCAAGGCTGGTC TGAAGAGCAGCCTCTTCCTA 700 TAGCAGAAAGGCTGGTCTTG CCTTGGAGACATGCCTCAGG 200 GAGACGGCCGCCGTGCCGCA TTCTCTTTGTTTCTCCCTCT 200 CCGTGCCGCCACCAGAGCGT GGTGTCGCTGAAGTTCCTCT 800 TCAGGTCGTTCGGTATCAGG GCTAGGTTTTGGTCCTTCGT 600 CGTCGGAGGCCTTGGTGCGT GGTTCAACGTCACCAAGGGT 1150 CGTCGGAGGCCTTGGTGCGA CAATGTAATCATCGAAGAGG 300 TGTACCACTCAAGACAAGGT AGGGCTAGGAAATTATCAAC 200 TTCTAACCATAATGGCACGA AACCTCAAAGGAAGCAAGGA 250 ATTAAGGATGCAGAGCAACT ACCTTCAAGTGTGGTGTCGT 250 AGAGCGTGCTCCCTGAGGCC AGACCTTCAAGTGTGGTGTT 250 AGAGCGTGCTCCCTGAGGCT CCTTGATCTTATCAGCATGA 300 TCCGCCCCTTCTCTTATGGG GAACAAGTGTAGATGAACAT 350 AGGGCAACGTGACCATCGCT AGTTGAGATGAACAGCACAG 500 CGTTTGGCTCTGGAGTCGTG GCGGCGCTCAAAGATCCTCC 600 CAGCCAGAGTGAAGCCATCA GCTTAACTTCTTCCACAGCA 450 ACTAGCTGCACAGGCCACTC GCTTAACTTCTTCCACAGCG 400 ACTAGCTGCACAGGCCACAA GAAGACTGTAAACATCTTAG 200 ACAAGAAGAAGAGGATCGCC TCTTGGCCATGCTCTCGCTG 300 TGAGGGGACCCAAGAGGGCC TACTCTCAAACTTTTCCCGG 500 ACAGCATCCCCATCAACCTC CGGCACATTCGTACTCTCAT 1000 TTGATGACAAGCTCCACCCA

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RESULTS AND DISCUSSION Results Primer design and evaluation Designing primers is an important and critical step with any PCR technique. In this study the primers were developed based on an SNP occurring in the EST alignment. The selected EST was aligned against other wheat ESTs pulled out from either wheat EST-NSF or ITEC databases to locate any SNPs. Twenty nucleotides were chosen upstream from the polymorphic nucleotide, including the polymorphic nucleotide, to construct forward primers. Reverse primers were constructed by translating 20 nucleotides downstream of the different polymorphic nucleotide from the same sequence. Before mapping the EST into wheat deletion lines, the primers were evaluated to amplify Chinese Spring wheat genomic DNA. Only the primers that produced a single band were used for further analysis. The primers were then used to determine the chromosomal location of ESTs using a series of Chinese Spring wheat nullisomic stocks (Sears, 1954). In general, each evaluated primer produced single band in all nullisomic-tetrasomic lines except in the one line where the EST was physically located. If the primers developed from the same EST family also produced a single PCR band with a different size, their chromosomal locations using nullisomic lines showed that those ESTs could be assigned to

different chromosomes from the same homologous group. For example, primers EST 51, 52, and 53 were developed from the ESTs originating from the same contig (DSW03C5_contig5798.3), and produced one band but were located on different chromosomes (1A, 1B, and 1D) (Table 2). Table 2. Blinded names, primer names, EST names, contig origin, location, and size of PCR products. Blinded Primer EST names Contig origin Location names names 51F/R F064 BM140481 DSW03C5_5798.3 1AL R391 BM137664 BM135238 BE500501 BE500167 BE498208 BF484824 BF484612 52F/R F076 BE202634 DSW03C5_5798.3 1BL R448 BF483613 BE498846 BE500808 BF482964 53F/R F278 BE500375 DSW03C5_5798.3 1DL R546 BE499726 BE499492 BF484674 BM137850 BE498130

Figure 1. An example of mapping EST 152 to chromosome 2D using PCR technique.

Size (bp) 175

300

380

CHIKMAWATI et al. â&#x20AC;&#x201C; PCR-based EST mapping of Triticum aestivum

Figure 2. An example of EST mapping on homoeologous chromosome group 2.

Physical mapping of ESTs The EST assignment to a deletion (Figure 1) was based on the presence or absence of the band among the series of deletion lines. A band that is missing in particular deletion line means the band is located to the chromosome region that has been deleted from that particular deletion line and has been assigned to a bin fraction length where the chromosome part is missing. We mapped 68 ESTs into assigned bins of the deletion lines. The blinded names of the primers, EST accessions, and assigned bins for 68 mapped ESTs are presented on Table 3. The majority of the ESTs were mapped to homologous chromosome group 2 (Figure 2), and the fewest were mapped to homologous chromosome group 6. All the long arms of the seven homologous chromosome groups were assigned by EST (50 ESTs), whereas not all of the short arms of chromosomes were assigned. There were 29, 23, and 16 ESTs that mapped into the three genomes A, B, and D, respectively (Table 4). Gene prediction of mapped ESTs We performed BLASTX (Basic Local Alignment Search Technique) analysis (Altschul et al., 1997) to establish what gene function that EST represented, and to compare the six-frame conceptual translation products of a nucleotide query sequence (both strands) against a protein sequence database. The

matching gene with the highest score, lowest E-value, and the same as or a higher than 90% identity was selected as the gene that represented the EST being analyzed. All the ESTs were assigned as a known gene from the database. Some ESTs with different accession numbers that were mapped either to the same homologous chromosome group or different chromosome represented the same gene. Many reports (Khlestkina et al., 2002; Clarke et al., 2003; Danyluk et al., 2003; Watanabe and Koval, 2003) have shown that most genes in hexaploid wheat can be found in triplicate with one copy in each genome. Some genes can also have additional copies in other chromosomes from the same or different homologous groups. Our results showed an agreement with the previous studies that 12 genes (46%) were mapped on at least two homologous chromosomes, while seven of them were specific to chromosome groups 2, 3, and 5 (Table 5). Among 26 mapped genes, 10 genes (38.5%) were mapped only to one chromosome, four genes (15.4%) were mapped to two homologous chromosomes, three genes (11.5%) were mapped to three homologous chromosomes, five genes (19.2%) were mapped to two or three homologous and other chromosomes, and the rest of the genes (15.4%) were randomly mapped, which means those genes were mapped into more than one different chromosome from a different homologous group (Figure 3).

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Table 3. Chromosome location, bin name, blinded name of primers, and EST accession numbers. Chromosome

Bin name

1 AL

1AL1-0.17-0.61

1 BL

1BL2-0.69-0.83

1 DL

1DL2-0.41-1.00

2 AL

C-2AL1-0.85

2 BL

2BL6-0.89-1.00 2BL2-0.36-0.50

2 DL

2DL3-0.49-0.76

2 AS

2 BS 2 DS 3 AL 3 BL 3 DL 3 AS 3 BS 3 DS 4 AL

C-2AS5-0.78 2AS5-0.47-1.00 C-2BS1-0.53 2DS5-0.47-1.00 3AL3-0.42-0.78 C-3AL3-0.42 C-3BL2-0.22 3DL2-0.27-0.81 C-3AS2-0.23 3BS9-0.57-0.78 3DS6-0.55-1.00 C-4AL12-0.43 4AL4-0.80-1.00

4 BL 4 BS

4AL5-0.66-0.80 4BL1-0.81-1.00 4BS1-0.81-1.00

5 AL

5AL10-0.57-0.78

5 BL 5 DL

5BL14-0.75-0.76 C-C5DL1-0.6 5DL-0.76-1.00

5 AS 5 BS 6 AL 6 BL 7 AL 7 AS 7 BL 7 BS

5DL1-0.6-0.74 5AS3-0.75-0.98 5BS6-0.81-1.00 C-5BS4-0.43 5BS5-0.71-0.81 C-6AL4-0.55 C-6BL3-0.36 7AL21-0.74-0.86 7AL18-0.90-1.00 7AL1-0.39-0.71 7AS1-0.89-1.00 C-7BL2-0.33 7BS1-0.27-1.00 C-7BS1-0.27

Blinded name 51 231 302 52 141 53 301 103 133 153 201 223 251 311 102 132 151 221 222 253 101 131 152 224 422 32 182 161 42 181 81 421 82 172 83 261 412 411 54 431 142 331 84 55 56 432 322 401 402 11 273 283 31 321 403 61 271 62 281 272 21 22 301 91 211 333 292 143 361

EST accession BE500501 BQ619858 BM138684 BF483613 BM134418 BE500375 BE499039 BF484255 BF202081 BM138609 BF202965 BF202198 BG907231 BG904954 BM140371 BF202081 BE500314 BQ620117 BE500314 BG904361 BM140580 BM137809 BM138609 BF482340 BQ238323 BF483165 BQ240701 BE499318 BE500396 BQ240701 BF483171 BE425039 BE499999 BE423540 BF484421 BG909863 BG905429 BG905429 BF482753 BQ620599 BM134418 BG907633 BF484421 BF482680 BF482845 BQ237808 BE424825 BE424633 BG904428 BE500218 BQ620354 BM140514 BF202379 BG906277 BG904212 BE637755 BF202040 BF482523 BG906628 BF482437 BF201679 BF202619 BE499039 BE498885 BE499290 BG910130 BE422799 BE499017 BG906045

Table 4. Distribution of the ESTs in each wheat chromosome group. Chromosome groups 3 4 5 6 L* S* L S L S L S L S L S A 3 0 7 3 2 1 4 0 2 2 1 0 B 2 0 6 1 2 1 1 3 1 2 1 0 D 2 0 5 1 1 1 0 0 6 0 0 0 Total 7 0 18 5 5 3 5 3 9 4 2 0 Note: * L= long arm. S= short arm. Genome

7 L 3 1 0 4

S 1 2 0 3

Table 5. Distribution of mapped genes in wheat genome. Genes

Chromosome location

Alpha-tubulin Chlorophyll a/b binding protein Cold shock protein-1 Histone H3 Plasma membrane P-type proton pump ATPase S-adenosylmethionine decarboxylase Malate dehydrogenase (MDH) DNA Binding Protein S1FA Ribosomal protein s6 RPS6-2 Elongation Factor-1 alpha Sucrose synthase type 2 Translation initiation factor 5A Putative ribosomal protein L18a, cytosolic NADP-specific isocitrate dehydrogenase Putative 40S ribosomal protein S3 NADP-dependent malic enzyme Lhcb6 protein (Lhcb6) Ribosomal protein S29-like protein UDP-glucose pyrophosphorylase Lipid transfer protein 7a2b Thioredoxin M Methionine synthase protein Putative 60S ribosomal protein L6 High mobility group protein Expressed protein RL5 ribosomal protein

1AL, 1BL, 1DL, 4AL, 4BS 1AL, 2AL, 2BL, 4AL, 4BS 1AL, 1DL, 7AL 1BL, 2BS, 4BL, 6AL, 7BS 2AL, 2BL, 2DL 2AL, 2BL, 2DL 2AL 2AL 2AS, 3AL 2AS, 5DL 2AS, 2DS 2AS 3AL, 3BL, 3DL, 4BL 3BL, 7BS 3AS 3BS, 3DS 4AL 5AL, 5DL 5AL, 5BL, 5DL 5DL 5DL, 5BS, 7BS 5AS, 5BS 6 BL 7AL 7AL 7BL

Mapped only in one chromosome

15.4%

Mapped in two homoeologous chromosome

19.2%

38.5%

11.5% 15.4%

Mapped in three homoeologous chromosome Mapped in homoeologous chromosome and other chromosomes Mapped randomly more than one chromosome

Figure 3. Genes distribution on wheat genome.

CHIKMAWATI et al. â&#x20AC;&#x201C; PCR-based EST mapping of Triticum aestivum

Comparisons between PCR and RFLP-based EST mapping The reliability of PCR-based EST mapping was evaluated by comparing EST maps generated from PCR techniques to RFLP techniques. The RFLPbased mapping was performed by hybridization of cDNA clones that were used to generate ESTs. Using the PCR-based approach, before hybridization, the ESTs were screened to select singletons, which are ESTs that only represent single gene. The selected singleton was then hybridized with the EcoRIdigested genomic DNA from all wheat nullisomic, ditelosomic, and deletion lines. Bin assignment procedure was the same as in the PCR-based approach. The EST mapping data are publicly available through the wheat EST-NSF database (http://wheat.pw.usda.gov/westsql). We performed an EST search based on the gene names listed in the Table 5 in order to compare both maps. Any resulting ESTs, including EST accession number and bin assignment, were compared to the corresponding EST from the PCR-based EST map and mRNA from the gene to evaluate the homology and the bin location for the two ESTs. We identified three groups of genes. First, eleven genes were mapped in the same bin. Although the EST accession numbers were different, sequence comparison between each pair of ESTs showed high identity. In the case where no homology was observed, both ESTs showed high homology to the same gene at different places indicating both ESTs originated from the same gene. Second, three genes were mapped to different bins, but on the same chromosome. Experimental errors probably can explain this situation, such as incorrect deletion lines used, poor hybridization, or gene duplications and translocations. Poor hybridization could lead to the misinterpretation when assigning the EST to a particular bin. The last group was a group of 12 genes that could not be found in the RFLP-based EST map. This is probably because those genes have not been mapped with RFLPs.

Discussion Mapping ESTs to hexaploid wheat is aimed mainly at revealing the structure and function of the hexaploid wheat genome; however, it is a laborious and expensive task. Fortunately, wheat has a set of aneuploid stocks including nullisomic-tetrasomic, ditelosomic, and deletion lines (Sears, 1954; Endo and Gill, 1996) that have been widely used in physically mapping and genetic analysis of wheat. Conventional mapping approaches using RFLPs sometimes cannot be avoided, even though it is expensive, laborious, time consuming, and includes risks associated with radioisotopes. Expressed sequence tag mapping using PCR is a good alternative approach because it offers a cheap, fast, safe, and reliable approach. The successful key to

any PCR technique is in designing primers that can amplify genomic DNA consistently, resulting in the expected PCR product size and polymorphic bands. In this research, PCR primers were developed from wheat ESTs, and mapped to the hexaploid wheat genome using a series of deletion lines. Bin mapping of ESTs to hexaploid wheat using PCR requires a specific approach in constructing the primers. The primers should amplify a gene from only one chromosome. Therefore, to map a gene in hexaploid wheat with one copy from each homologous chromosome requires three different primers combinations derived from the same gene. With, an assumption that each homologous chromosome is derived from a different parental genome, the same gene, derived from each homologous group, will have at least one SNP in its coding region, which can be recognized through the alignment of EST sequences of those genes. To ascertain the amplification will occur only in one homologous chromosome, three strategies were employed when primers were designed. First, the primers were designed from the EST that has been aligned with at least three wheat ESTs showing at least one SNP in every two ESTs. Second, the primers were designed from the polymorphic nucleotide site where the polymorphic nucleotide was in the 3â&#x20AC;&#x2122; end of primers to ensure that one primer combination will amplify one EST, but not the other ESTs. Finally, at least three combinations of the primers were designed from different places in the coding region specifically to produce different amplification sizes. This strategy was used to avoid one PCR product from one chromosome being confounded by another PCR product from the other homologous chromosome. We mapped 68 ESTs that represented 26 genes into all seven homologous chromosome of wheat. On averages, more than two ESTs or primer combinations were required to map a gene in hexaploid wheat. Comparative analysis between the EST map from this study and the EST map based on RFLPs revealed that about 14 genes that have been mapped using both approaches were mapped on the same arm of the same homologous chromosome. This result indicated that the PCR-based EST mapping, including the method for designing the primers, was a reliable approach to map ESTs in hexaploid wheat. We also observed that five genes had a different copy number between the two maps. Since the PCRbased EST mapping technique was aimed to map single band on every homologous chromosome, it was possible that with a particular gene, we could develop one pair of primers that only mapped to one chromosome. On the other hand, the RFLP-based mapping approach used one probe for a particular gene, and could produce more than one hybridization band, which was designated as locus, that could be mapped into more than one chromosome.

B I O D I V E R S I T A S Vol. 10, No. 2, April 2009, pp. 55-62

Beside the advantages of this PCR technique for mapping ESTs in hexaploid wheat, there was constraint that needs to be overcome in order to improve this technique. To map a gene to hexaploid wheat requires at least three properly working primer combinations, which is not easy to obtain and is time consuming.

CONCLUSION Sixty eight ESTs were successfully mapped into assigned bins of wheat deletion lines. The majority of the ESTs were mapped to homologous chromosome group 2, and the fewest were mapped to homologous chromosome group 6. There were 29, 23, and 16 ESTs mapped into the three genomes A, B, and D, respectively. The 68 mapped- ESTs represented 26 genes, and 14 of them were mapped to the same homologous chromosomes on the EST map, based on RFLPs. Twelve genes (46%) were mapped on at least two homologous chromosomes, while seven of them were specific to chromosome groups 2, 3, and 5. This study demonstrated that the PCR technique, including the development of primers, was reliable for mapping ESTs in hexaploid wheat.

ACKNOWLEDGEMENT The research is part of the dissertation written by the first author. We thank to Dr. Daryl Somers for providing primer sequences and NSF wheat EST mapping Project for the wheat Addition and Nullisomic lines.

REFERENCES Altschul, S.F., T.L. Madden, A.A. SchĂ¤ffer, J. Zhang, Z. Zhang, W. Miller, and D.J. Lipman. 1997. Gapped BLAST and PSIBLAST: a new generation of protein database search programs. Nucleic Acids Research 25: 3389-3402.

Cato, S.A., R.C. Gardner, J. Kent, and T.E. Richardson. 2001. A rapid PCR-based method for genetically mapping ESTs. Theoretical and Applied Genetics 102: 296-306. Clarke, B. C., T. Phongkham, M. C. Gianibelli, H. Beasley, and F. Bekes. 2003. The characterisation and mapping of a family of LMW-gliadin genes: Effects on dough properties and bread volume. Theoretical and Applied Genetics 106: 629-635. Danyluk J., N. A. Kane, G. Breton G., A.E. Limin, D.B. Fowler, and F. Sarhan. 2003. TaVRT-1, a putative transcription factor associated with vegetative to reproductive transition in cereals. Plant Physiology 132: 1849-1860. Endo, T. R., 1988. Induction of chromosome structural changes by a chromosome of Aegilops cylindrical L. in common wheat. Journal of Heredity 79: 366â&#x20AC;&#x201C;370. Endo, T.R., and B.S. Gill. 1996. The deletion stocks of common wheat. Journal of Heredity 87: 295-307. Fischer, P.J., and L.S. Lerman, 1983. DNA fragments differing by single base-pair substitutions are separated in denaturing gradient gels: correspondence with melting theory. Proceedings of the National Academy of Sciences USA 80: 1579-1583. Harry, D.E., B. Temesgen, and D.B. Neale. 1998. Codominant PCR-based markers for Pinus taeda developed from mapped cDNA clones. Theoretical and Applied Genetics 97: 327-336. Khlestkina, E., E.G. Pestsova, E. Salina, M.S. Roeder, V.S. Arbuzova, S.F. Koval, and A. Boerner. 2002. Genetic mapping and tagging of wheat genes using RAPD, STS and SSR markers. Cellular and Molecular Biology Letters 7: 795-802. Messing, J., and J.V. Llaca. 1998. Mapmaker: an interactive computer package for constructing primary genetic linkage maps of experimental and natural populations. Genomics 1: 174-181. NSF database (http://wheat.pw.usda.gov/westsql). Orita, M., Y. Suzuki, T. Sekiya, and K. Hayaki. 1989. Rapid and sensitive detection of point mutations and DNA polymorphisms using the polymerase chain reaction. Genomics 5: 874-879. Saghai-Maroof, M.A., R.M. Biyashev, G.P. Yang, Q.Zhang, and W. Allard. 1994. Extraordinarily polymorphic microsatellite DNA in barley: species diversity, chromosomal locations, and population dynamics. Proceedings of the National Academy of Sciences USA 91: 5466-5470. Saier, M.H. 1998. Genome sequencing and informatics: new tools for biochemical discoveries. Plant Physiology 117: 1129-1133. Sears, E.R. 1954. The aneuploids of common wheat. Research Bulletin 572: 1-58. Missouri: University of Missouri Agricultural Experiment Station. Watanabe N and S.F. Koval. 2003. Mapping of chlorina mutant genes on the long arm of homologous group 7 chromosomes in common wheat with partial deletion lines. Euphytica 129: 259265.

BIODIVERSITAS Volume 10, Number 2, April 2009 Pages: 63-69

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100202

Genetic Transformation of Transcription Factor (35S-oshox4) Gene into Rice Genome and Transformant Analysis of hpt Gene by PCR and Hygromycin Resistance Test 1,♥

ENUNG SRI MULYANINGSIH , REGY HERMAWAN , INEZ HORTENZE SLAMET-LOEDIN

Research Centre for Biotechnology, Indonesian Institute of Sciences (LIPI), Cibinong-Bogor 16911 Department of Biochemistry, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University (IPB) Bogor 16680. Received: 8 th November 2008. Accepted: 20th December 2008.

ABSTRACT Global warming, climate change and crop extensification in marginal dryland areas are related to long dry season and water deficit. The water availability is an important factor in improving plant production. Application of drought tolerant rice cultivars is one of several options that might be used. Genetic engineering at the level of transcription factors is particularly promising strategy to develop drought tolerant rice varieties. Transcription factors regulate a wide range of target genes in which of them contribute to stress tolerance. HD Zip genes are transcription factor that potential in the adaptation of plants to some environment stresses including water deficit. HD-ZIP oshox4 (oryza sativa homeobox) gene controlled by 35S promotor is inserted into pCAMBIA 1300 vector with hpt (hygromycin) gene as a selectable marker. The aim of this research is to obtain transgenic rice plant from transformation with 35S-oshox4 plasmid, segregation analysis of marker gene (hpt) by PCR method at T0 and T1 generation, and hygromycin resistance analysis of seeds. Recombinant plasmid was transformed into rice genome of IRAT 112 and rojolele cultivars using Agrobacterium tumefaciens. The results showed that transformation efficiency of IRAT 112 was 5.7-13.6% and 26-66,7% for rojolele. While regeneration efficiency for IRAT 112 is 4.7-43.7% and 23-44.1% for rojolele. The result of hygromycin resistance test at T1 seeds were obtained 14 lines cv. rojolele segregation Mendelian for hpt gene. The PCR analysis using specific primers for hpt gene at the parent (T0) from 14 lines showed that 7 lines contain the gene. At the second generation (T1), PCR analysis using hpt primers showed that 3 from 4 lines were followed Mendelian segregation pattern by the presence of specific band. © 2009 Biodiversitas, Journal of Biological Diversity Key words: HD-Zip oshox (oryza sativa homeobox), 35S-oshox4, hpt, Agrobacterium tumefaciens, PCR.

INTRODUCTION Global warming, unpredictable climate change and crop extensification in marginal dryland area are related to long dry season and deficit water. Water availability is a major limiting factor for increase of food production. According to Gorantla et al. (2005), rice crop consume about 90% from total water that is utilized for agricultural need at Asian. About 80% of total rice crop need water because most of rice crops are irrigated rice field (55%) and rain field (25%). Water deficit caused stress for plant. Water deficit will be major problem which influence global productivity. The drought stress effect could be lost of productivity until more than 70% (Bray et al., 2000). In attempt to dissolve of that problem, drought tolerance plant was used. Drought tolerance plant

 Corresponding address: Jl. Raya Bogor Km.46 Cibinong-Bogor 16911 Tel. +62-21-8754587; Fax. +62-21-8754588 e-mail: enungf@yahoo.com

could be obtained by some technique, such as genetic transformation and plant breeding. The important thing in creating this plant is understanding drought tolerant. Drought tolerance character is encoded by many genes. Shinozaki and Yamaguchi Shinozaki (1997, 2007) classified this into two groups; first, protein that most probably function in drought (protection factors of macromolecules, enzymes for osmolyte biosynthesis, detoxification enzymes, water channel, transporter); and second is regulatory protein (protein kinase, protein phosphatase, enzyme phospholipid metabolism and transcription factors). Genetic Transformation at transcription factor level will enable to obtain of drought tolerant rice, because transcription factor regulate other genes that responsible to drought tolerance. Some of transcription factors have been characterized, for example DREB (dehydration responds element binding) (Yamaguchi Shinozaki and Shinozaki, 2001; Sakuma et al., 2006), SNAC (stress responsive NAC1) (Hu et al., 2006), and HD Zip (Homeodomain leucine zipper) (Meijer et al., 1997; Deng et al., 2002). The over expression of these gene in some plants

B I O D I V E R S I T A S Vol. 10, No. 2, April 2009, pp. 63-69

caused increase of drought tolerance (Scarpella et al., 2005; Hu et al., 2006). HD Zip genes were reported to be related with adaptation and development of plants under stressful environment. HD Zip genes in rice plant are classified into family I, II, III that consist of 33 HD Zip genes oshox (oryza sativa homeobox) which is spread in 12 chromosomes. There are only two genes oshox have been identified which is oshox1 (HD Zip II) and oshox4 (HD Zip I) that regulate by drought stress. The expression of genes are up and down regulated under drought situation and depend on plant sensitivity to drought (Purwantomo, 2007; Agalou et al., 2008). Research Centre for Biotechnology LIPI have obtained first generation (T0) of putative transgenic rice from rojolele and IRAT 112 cultivars that transformed with transcription factor HD Zip oshox4 gene. This gene is controlled with constitutive promotor CaMV 35S (cauliflower mozaic virus). Cassette 35S oshox4 and terminator were inserted in pCAMBIA 1300 plasmid on HindIII site. Hygromycin phosphotransferase (hpt) gene to generate hygromycin antibiotic resistance for transformant selection. The recombinant plasmid was designated as 35S oshox4 and transformed into javanica cultivar (rojolele) and indica cultivar (IRAT 112) using Agrobacterium tumefaciens (A. tumefaciens). The aim of research is to obtain transgenic rice plant transformed with 35S-oshox4 plasmid, to evaluate lines containing hpt gene based on PCR analysis, to evaluate expression and segregation pattern of hpt using bioassay hygromycin seeds.

MATERIALS AND METHODS Rice cultivar of javanica rojolele and indica IRAT 112 transformed by 35S-oshox4 plasmid were made available Dr. A.H Meijer and Dr Pieter B.F. Ouwerkerk (Institute of Molecular Plant Science, Leiden University). Strain A. tumefaciens using EHA 105 resistance to rifampycin antibiotic. Analysis transformant at first (T0) and second (T1) generation plant population that property RC Biotechnology LIPI. Materials for DNA isolation comprise: isolation buffer [lysis buffer (Tris HCl pH 7.5 0.2 m, EDTA 0.05 M, NaCl 2 m, and CTAB 2%)]; extraction buffer [extraction buffer (sorbitol 0.35 m, Tris HCl pH 7.5 0.1 m, and EDTA 5 mM); sarkosil 5%], N2 liquid, chloroform: isoamylalcohol (24:1), isopropanol, ethanol 70%, TE buffer (Tris HCL pH 8.0 10 mM and EDTA PH 8.0 1 mM). Material used for PCR reaction: plant DNA, PCR buffer, dNTPs, primary gos-5 forward and reverse (5’CCGACCTCGAGGACATCGG CAACAG 3’) and (5’ GCCGAGAGCAGCAGGAACTT GACAGG 3’), primary hpt forward and reverse (5’ GATGCCTCCGCTCGAAGTAGCG 3’) and (5’ GCATCTCCCGCCCGTGCAC 3 ’), dH2O, agarose gel and Tris Boric Acid EDTA buffer (TBE). Material those

were utilized for hygromycin test: 50 mg/mL hygromycin, sterile aquadest, sterile filter paper, alcohol 70%, benlate 3%, and NH4ClO 2%. Genetic transformation into rice genome Recombinant plasmid 35S-oshox4 was inserted into A. tumefaciens strain EHA 105 by electroporation method. Bacteria were cultured in solid AB medium which contained rifampycin 20 mg/L and kanamycin 50 mg/L. Bacteria culture were then incubated at 28C for 3 days. This culture was used for cocultivation. Transformation into embriogenic callus using A.tumefaciens was perfomed following methods as described elsewhere (Hiei et al., 1994). A. tumefaciens was subcultured in AAM liquid medium without antibiotic and shaked to 1-2 hours until OD600: 0,4-0,5. The embryogenic callus from cv. rojolele and cv. IRAT 112 were induced in IK3 medium (LS media that contains 2,4-D 2,5 mg/L and solidified with phytagel 0,2%) (Slamet-Loedin et al., 1997). Callus induction was done for 2 weeks in dark room. For infection, callus is soaked in A. tumefaciens culture up to 30 minutes. Callus and bacteria at co-cultivation in IK3-AS medium (IK3 contain of acetosyringone 100 mM) and incubation on 25C for 3 days in dark. After co cultivation, callus washed by cefotaxime 400 mg/L and cultured at IK3C250 H50 as selection medium (IK3 contain of cefotaxime 250 mg/L and hygromycin 50 mg/L) for 2 weeks. Callus were subculture at second selection medium for two weeks IK3C50 H50. The resistance callus to hygromycin were subculture into R3B medium for regeneration (LS + IAA 0,5 mg/L + BAP 0,3 mg/L and phytagel 0,5%) (Slamet-Loedin et al., 1997). Obtained plantlets were moved into MS medium without hormone. The normal plantlets were acclimatized into soil medium at greenhouse. DNA isolation for PCR DNA Isolation for PCR from leaf (2-3 weeks plant). Isolation method as follows: leaf 5 cm length was inserted put into microtube 1.5 mL, add with N2 liquid then grinded and added with 750 L isolation buffer. The mixtures were then incubated at 65oC for 1 hour. Later tube is added 750 L chloroform: isoamylalchohol (24:1) and centrifuge 5 minutes at 12.000 rpm, 4oC. The supernatant was transferred into new tube and added with 400 L isopropanol (cold), then centrifuge for 6 minutes at 12.000 rpm on 4oC. Supernatant was discarded and pellet was washed by ethanol 70% then centrifuge at 12.000 rpm on 4oC for 3 minutes. Supernatant is discarded and pellet in tubes dried. Palette from DNA was dissolved by TE 50 L and stored at 20oC. PCR analysis at T0 plant Plants DNA were obtained from T0 and T1 population. PCR reaction is enclosed with primer gos5 I as internal rice gene. Negative control using DNA plant controls (without transformation) and water,

MULYANINGSIH et al – Genetic transformation of transcription factor

RESULT AND DISCUSSION Transformation into rice genome The transformation result into genome of two cultivar rice is presented at Table 1. Based on this

IRAT112 Rojolele

469 840 60 923 584

3 21 15 106 49

13,6 5,7 66,7 26,0 36,5

Regeneration efficiency (%)

64 48 40 240 213

Transformation efficiency (%)

PCR analysis of T1 plant The normal seedling based on hygromycin test and segregation follows Mendelian (base on chi square) were planted on soil medium at greenhouse. Thirty seeds for each line were planted. DNA template for PCR reaction was isolated from leaf derived from 2-3 weeks old seedling according to the method described previously. PCR analysis was performed to confirm the presence of hpt gene on second generation plant (T1). PCR conditions were the same with T0 analysis plant.

Table 1. Summary of transformation using of recombinant plasmid 35S-oshox4 on cv. IRAT 112 and rojolele.

Number of regenerated callus

O i = phenotype’s observing point goes to i. E i = phenotype’s expectation point goes to i.

Number of resistance hygromycin callus

2 = (O i – E i) 2 Ei

Number of transformed callus

Hygromycin test As many as 76 lines rice seed T1 (planted 50 seeds for each line). Seeds was husked, washed with sterile aquadest and followed with alcohol 70% for 1 minute and washed with sterile aquadest. Seeds were then soaked with benlate 3% solution for 30 minutes and washed with sterile distilled water until clear. Finally, seeds were washed by 2% NH4ClO for 30 minutes and rinsed with sterile distilled water for 5 minutes 10 times repeatedly at laminar air flow. Planted seed on sterile filter paper already been damped by 50 mg/L hygromycin. As for control, filter paper was just soaked with distilled water. Seed were placed in dark room to stimulate germination for 3 days, then moved under lamplight. The observation for seedling growth was carried out 2 weeks after plantation under following classification: normal seedling, abnormal and not grown (total number of normal seedling as amount of plant individual that using chi square to observe segregation pattern hpt gene). With opportunity point 5% and degree of freedom 1, therefore point chi square shall be smaller than F table (3,84). It means pattern of segregations follow Mendelian which is 3: 1. Equation for tests chi square as follows:

table transformation efficiency and regeneration of cv. rojolele was higher than that of cv. IRAT 112. The rojolele cultivar was relatively easy to form embryogenic callus than IRAT 112. Successful genetic transformation is related with embryogenic callus formation and plant regeneration system. According to Ge et al. (2006) callus induction and regeneration in rice tissue culture depend on a number of factors, such as genotype of the donor plant, type and physiology status of the explants, composition and concentration of the basal salt and organic components, and plant growth regulators in the culture medium. Among these factors, genotypic difference is the most important. IRAT 112 Cultivar consists of indica rice. Most of the indica varieties belong to the group I, which these varieties have been quite recalcitrant cultivars (difficult for regeneration and transformation) in tissue culture and genetic transformation (Wunn et al., 1996; Zhang et al., 1998). Even success of transformation in indica rice was reported, but the results showed either low transformation efficiency or success only with very specific genotype (Zhang et al., 1997; Lin and Zhang, 2005). Therefore low efficiency and regeneration on IRAT 112 suggest the genotype of plant. The lower transformation efficiency on this genotype also occur in other cultivars such as IR72 and IR 64 each by level efficiencies 4.2% and 2.5 10.1% (Aldemita and Hodges, 1996; Hiei et al., 2006; Khanna and Raina, 2002). Although recently reported that the transformation efficiency in ten indica rice cultivars could be obtained until 30% for each immature embryo with optimation things (Hiei et al., 2006). In the meantime on rojolele cultivar transformation efficiency and regeneration were higher than IRAT 112 because rojolele belong to javanica rice. This genotype (javanica) is easier for transformation and regeneration and is not a recalcitrant type.

Cultivars

while positive control uses 35S-oshox4 plasmid. PCR reaction mixtures were as follows: 1x PCR buffer, dNTP 0.05 mM, Taq Polymerase 0.05 u, primer 2. 5 ng/µL gos-5 reverse and forward, primer 2.5 ng/µL hpt reverse and forward. PCR temperature and cycle it one denaturation cycle (95oC, 10 minutes): 40 amplification cycles [denatures 95oC 1 minute, annealing 55oC 1 minute, synthesis 72oC 1 minute]; 72oC 10 minutes (final elongation); 4oC (store).

4,7 43,7 37,5 44,1 23,0

Hygromycin test on T1 generation Hygromycin test on seed (the seed from T0 plant) conducted to select a lot of number from lines putative transgenic. This analysis will reduce economic cost. The selection based on selectable

B I O D I V E R S I T A S Vol. 10, No. 2, April 2009, pp. 63-69

PCR analysis of T0 plant The aim of PCR analysis for hpt gene was to evaluate integration of the selectable marker and the segregation based on number of integrated plants. The 14 lines plant with Mendelian segregation based on hygromycin test from the second generation (T1) were planted in soil medium in green house with 30 seedlings for each line. Parallel with this activity, PCR analysis was done for the 14 plant as the parent (T0).

Rojolele T1.R.B.16.6 T1.R.B.17.1 T1.R.B.17.2 T1.R.B.17.3 T1.R.B.17.4 T1.R.B.18.1 T1.R.B.19.1 T1.R.B.26.1 T1.R.B.28.1 T1.R.B.29.1 T1.R.B.29.2 T1.R.B.29.3 T1.R.B.29.4 T1.R.B.30.1 T1.R.C.2.1 T1.R.C.2.4 T1.R.C.2.7 T1.R.C.2.8 T1.R.C.2.9 T1.R.C.2.10 T1.R.C.2.11 T1.R.C.2.12 T1.R.C.2.13 T1.R.C.2.15 T1.R.C.2.16

12 15 7 38 34 41 22 27 35 28 5 14 14 20 2 19 41 20 19 35 27 30 26 30 38

64.00 49.00 99.23 0.03 0.82 2.78 23.68 9.00 0.33 8.33 103.84 53.78 61.50 30.54 131.44 34.29 1.97 30.54 29.96 0.11 10.35 4.00 14.11 6.00 0.17

3.84

T1.R.C.2.18 37 T1.R.C.4.1 26 19 34.29 T1.R.C.4.2 29 31 4.51 T1.R.C.4.3 19 21 27.00 T1.R.C.6.2 24 33 2.16 T1.R.C.7.1 28 32 2.46 T1.R.C.8.1 39 19 25.79 T1.R.C.8.2 22 20 28.44 T1.R.C.9.1 29 17 42.46 T1.R.C.10.1 13 8 84.26 T1.R.C.12.2 39 28 8.33 T1.R.C.13.1 43 33 1.53 T1.R.C.15.1 30 16 46.86 Control 7 rojolele Note: bold is lines with Mendelian segregation. Rojolele T1.R.A.1.5 T1.R.B.2.1 T1.R.B.4.1 T1.R.B.4.2 T1.R.B.6.1 T1.R.B.8.1 T1.R.B.8.2 T1.R.B.8.3 T1.R.B.8.4 T1.R.B.9.1 T1.R.B.9.3 T1.R.B.11.1

X table (Îą 0.05)

106.78 66.67 131.44 109.72 121.00 118.02 139.26 93.44 102.92 144.00 32.67 22.43 116.74 103.84 75.11 38.27 32.11 78.19 106.78 93.44 96.33 83.82 83.82 92.83 139.11

Chi square

5 12 2 5 3 3 5 7 6 0 20 23 4 5 10 18 19 9 5 7 7 9 9 8 1

Number of germinate seeds

IRAT 112 T1.I.B.1.1 T1.I.B.2.2 T1.I.B.2.3 T1.I.B.2.4 T1.I.G.1.1 T1.I.G.1.2 T1.I.G.3.1 T1.I.G.4.1 T1.I.G.5.1 T1.I.G.6.1 T1.I.G.7.1 T1.I.G.7.2 T1.I.G.7.3 T1.I.G.7.4 T1.I.G.7.5 T1.I.G.7.7 T1.I.G.9.1 T1.I.G.9.2 T1.I.G.9.3 T1.I.G.9.4 T1.I.G.9.5 T1.I.G.11.1 T1.I.G.12.1 T1.I.G.13.1 Control IRAT

Lines/ cultivar

Chi square

Table 2. Analysis of hpt gene expression on T1 generation seed which transform with oshox4 gene. Number of germinate seeds

marker could be an indicator for the presence other gene in the same of T-DNA. This examination is gone upon the expression of hpt gene as a selectable marker. With expression of hpt gene be provided that target gene oshox4 have also been integrated into rice genome. It is because hpt gene inserted on the same T-DNA with oshox4 gene (Figure 1). Hygromycin is an antibiotic resistance marker commonly used for genetic transformation on monocotyledon plant especially Gramineae (Bashir et al., 2004). This antibiotic hampered synthesis of protein by troubles translocation so causes error translation on ribosome 80S (Bashir et al., 2004). Hygromycin phosphotransferase enzyme obtained from hpt gene could detoxificate of hygromycin B antibiotic (Rodriguez and Nottenburg, 2003) and catalyze phosphorrylation of hydroxyl group in hygromycin antibiotic so that this antibiotic not active and not poison for plant cell (Brasileiro and Aragao, 2001). The analysis result in the second generation (T1) and lines used for hygromycin test were presented on Table 2. Water was used as control solution to observe seed germination from each line, whereas hygromycin solution as treatment solution. Seed which germinated and form normal seedling on hygromycin solution suggest that it may contain hpt expressed gene. Hygromycin was degraded by this plant, therefore it was not toxic and might not caused any trouble (Figure 2). Phenomenon of which hpt gene is integrated in the plant, genome but failed to express called gene silencing. There are three assumption of gene silencing which are cis inactivation, trans inactivation and co suppression. Cis-trans-inactivation occurred at transcription. Cis-inactivation occurs if 1 or many gene copies integrated on one locus in or near to genome sequent with high methylation. So, integrated gene will be methylation and not expression. Trans-inactivation occurred when insertion gene integrated on different locus and one of its integration experience cis- inactivation so becomes silencer to the homolog. Co- suppression happening on post transcription, processing, localization and or mRNA degradation while affluent mRNA production under strong promoter (Taylor, 1997). Other opinion was named of co-suppression occurred by involves coordinate silencing; sometimes coordinate reactivation from transgenic with endogenous homology or among 2 homolog transgenic (Matzke and Matzke, 1995). Base on this experiment, 14 lines of plant expressing hpt gene were obtained and the gene was segregated follows of Mendelian (3:1). These lines were from rojolele cultivar: T1.B.4.2, T1.B.6.1, T1.B.9. 3, T1.B.17. 3, T1.B. 17.4, T1.B.18.1, T1.B.28.1, T1.C.2.7, T1.C.2.10, T1.C.2.16, T1.C.2.18, T1.C.8. 1, T1.C.12.2, and T1.C.13.1. The absence of hygromycin resistance from IRAT 112 cultivar might suggest that integrated gene was difficult to obtain, or the inserted gene was not expressed. PCR analysis to evaluate parent containing hygromycin from IRAT 112 was not performed.

Lines/ cultivar

0.03 12.58 5.44 27.86 16.00 7.11 0.24 21.78 6.54 64.03 0.24 3.23 4.00 99.23

MULYANINGSIH et al â&#x20AC;&#x201C; Genetic transformation of transcription factor

EcoR EcoR I (1) Xma I (17) Ava I (17) Sma I (19) Bam HI (22) Pst I (44) Hind Hind III (44)

Ava I (1037)

lac z alfa

S 35 MV a C

Apa LI (7614) Hy gr r

Sta S1 pV

Apa LI (7312)

T-Border (Right) Ava I (407)

CS M

Ava I (7929) Sma I (7914) Ava I (7912) Xma I (7912)

Ava I (6835)

Ava I (2168)

pCAMBIA1300 8958 bp

POLY SITE

Ava I (2648)

T-Border (left)

p RE VS1 P -

n Ka

pBR 3 ori 22

Cla I (3472)

pBR322 bom site

Apa LI (4917) Apa LI (4419)

1197bp

2 x 35S P

450bp

685bp

HindIII

35S ter

oshox4 512bp

KpnI

500bp EcoRI

HindIII

Figure 1. The recombinant vector construction 35S Oshox4 is utilized on the research.

Figure 2. The hygromycin test at transformant seeds (T1). Resistance to hygromycin solution (A), resistance to solution without hygromycin (B), transformant susceptible to hygromycin solution (C), seeds control in hygromycin solution (D), seeds control in solution without hygromycin (E).

B I O D I V E R S I T A S Vol. 10, No. 2, April 2009, pp. 63-69

Figure 3. PCR result using hpt and gos-5 primers A. first generation (T 0), B. Second generation (T1).

Target gene to be amplificated is hpt and gos-5 with amplification product 429 and 231 bp respectively. According to Meijer et al. (1991) one of gene in rice genome, so called gos-5 can be served as internal control. Internal control is subjected to confirm that DNA used fine. Proper DNA and PCR reaction were expected to give a 231 bp length of PCR product in all samples including control (non transformation). The specific primer(s) was designed for amplification of specific gene(s). PCR analysis using hpt and gos-5 primers were done on 14 lines plant at first generation (T0). The analysis result showed that 7 lines plant T0 contain hpt gene and amplification product of gos-5 were present in all lines tested. Those are T0.C.4.2, T0.C.4.3, T0.C.8.1, T0.C.9.1, T0.C.12.2, T0.C.13.1, and T0.C.15.1. The result of PCR analysis on 14 lines (T0) was presented on Figure 3A. The PCR result at second generation (T1) using hpt primers was done for 4 lines which were T1.C.4.2, T1.C.4.3, T1.C.8.1 and T1.C.13.1. Tree of four lines were Mendelian segregation based on chi square tested. Those lines were T1.C.8.1, T1.C.13.1 and T1.C.4.2. The Mendelian segregation means that hpt was integrated in rice genome and inherited on its generation. PCR result was presented on Figure 3B.

CONCLUSION The transformation and regeneration for IRAT 112 callus cultivar using the recombinant plasmid 35S Oshox4 is lower than that of rojolele. Transformation efficiency for IRAT 112 is 5.7-13.6% whereas 2666.7% for rojolele. Regeneration of IRAT 112 callus between 4.7-43.7% and 23- 44.1% for rojolele. Fourteen lines of rojolele cultivar at second generation (T1) were Mendelian segregation (3:1) for hpt gene. The lines are T1.B.4.2, T1.B.6.1, T1.B.9.3, T1.B.17 3, T1.B.17.4, T1.B.18.1, T1.B. 28.1, T1.C.2.7, T1.C.2.10, T1.C.2. 16, T1.C.2.18, T1.C.8. 1, T1.C. 12. 2 and T1.C.13.1. PCR analysis using hpt primers for 14 lines parents (T0) showed that 7 lines contain hpt genes. The lines are T0.C.4.2, T0.C.4.3, T0.C.8.1, T0.C.9.1, T0.C.12.2, T0.C.13.1, and T0.C. 15.1. Three of 4 lines at second generation (T1) were Mendelian segregation based on PCR analysis indicated by the presence of specific band for hpt gene. The third lines are T1.C.4.2, T1.C.8.1, and T1.C.13.1. Integration of hpt gene as selectable marker in the genome suggest that oshox4 gene is also integrated into genome because both genes are from the same of T-DNA.

MULYANINGSIH et al – Genetic transformation of transcription factor

ACKNOWLEDGEMENT The authors thank to Dr. A.H Meijer and Dr Pieter B.F. Ouwerkerk for providing recombinant plasmid, Thank to Dr. Sigit Purwantomo and Dr. Satya Nugroho for discussion, advice, and financial support.

REFERENCE Agalou, A., S. Purwantomo, E. Overnas, H. Johannesson, X. Zhu, A. Estiati, R.J. de Kam, P. Engstom, I.H. Slamet-Loedin, Z.Zhu, M. Wang. L. Xiong, A.H. Meijer, and P.B.F. Ouwerkerk. 2008. A genome wide survey of HD-Zip genes in rice and analysis of drought responsive family members. Plant Molecular Biology 66: 87-103. Aldemita, R.R. and T.K. Hodges. 1996. Agrobacterium tumefaciens-mediated transformation of Japonica and Indica rice varieties. Plant. 199: 612-617. Bashir, K., M. Rafiq, T. Fatima, T. Husnain, and S. Riazuddin. 2004. Hygromycin based selection of transformants in a local inbred line of Zea mays (L). Pakistan Journal of Biological Sciences 7: 318-323 Brasileiro, A.C.M., and F.J.L. Aragao. 2001. Marker genes for in vitro selection of transgenic plants. Journal of Plant Biotechnology 3: 113-121. Bray, E.A., J. Bailey-Serres, and E.Weretilniyk. 2000. Response to abiotic stresses. In: Gruissem, W., B.B. Buchannan, and R. Jones (eds.). Biochemistry and Molecular Biology of Plants. Rocvkville: American Society of Plant Physiologists. Deng, X., J. Philips, A.H. Meijer, F. Salamini, and D. Bartels. 2002. Characterization of five novel dehydration responsive homeodomain leucine zipper genes from resurrection plant Craterostigma plantagineum. Plant Molecular Biology 49: 601-610. Ge, X., Z. Chu, Y. Lin, and S. Wang. 2006. A tissue culture system for different germplasms of indica rice. Plant Cell Report 25: 392-402. Gorantla, M., P.R. Babu, V.B.R. Lachagari, A.F. Feltus, and A.H. Peterson. 2005. Functional genomics of drought stress response in rice: Transcript mapping of annotated unigenes of an indica rice (Oryza sativa L. cv. Nagina 22). Current Science 89: 469-514. Hiei, Y. and T. Komari. 2006. Improved protocols for transformation of Indica rice mediated by Agrobacterium tumefaciens. Plant Cell Tissue and Organ Culture 85: 271-283 Hiei, Y., S. Otha, T. Komari and T. Kumashiro. 1994. Efficient transformation of rice (Oryza sativa L) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant Journal 6: 271-282. Hu, H., M. Dai, J. Yao, B. Xiao, X. Li, Q. Zhang and L. Xiong. 2006. Over-expressing a NAM, ATAF and CUC (NAC) transcription factor enhances drought resistance and salt tolerance in rice. PNAS 103: 12987-12992 Khanna, H.K. and S.K. Raina. 2002. Elite indica transgenic rice plants expressing modified cryIA (c) endotoxin of Bacillus thuringiensis show enhanced resistance to yellow stem borrer (Scirpophaga incertulas). Transgenic Research 11: 411-423.

Lin, Y.J. and Q. Zhang. 2005. Optimising the tisuue culture conditions for high efficiency transformation of indica rice. Plant Cell Report 23: 540-547. Matzke, M.A. and A.J.M. Matzke. 1995. How and why do plants inactivate homologous (trans) gene?. Plant Physiology 107: 679-685. Meijer, A.H., E. Scarpella, E.L. Van Dijk, L. Qin, A.J.C. Taal, S. Rueb, S.E. Harrington, S.R. McCouch, R.A. Schiperoot, and J.H.C. Hoge. 1997. Transcriptional repression by oshox1, a novel homeodomain leucin zipper protein from rice. Plant Journal 11: 263-276. Purwantomo, S. 2007. Karakterisasi Faktor Transkripsi HD-Zip untuk Toleransi Kekeringan dan Introduksi Gen-gen Penyandi Lintasan Biosintesis Asam Salisilat untuk Ketahanan Penyakit Blas pada Tanaman Padi. [Disertasi]. Bogor: Institut Pertanian Bogor. Rodriguez, R.C., and C. Nottenburg. 2003. Antibiotic Resistance Genes and their Use in Genetic Transformation Especially in Plants. CAMBIA. http:www.cambiaip.org/whitepapers/Transgenic/Ab_resistance/ books/ whole.pdf Sakuma, Y., K. Maruyama Y. Osakabe Y, M. Seki, K. Shinozaki, and K. Yamaguchi-Shinozaki 2006. Functional analysis of an Arabidopsis transcription factor, DREB2A, involved in drought responsive gene expression. The Plant Cell 18: 1292-1309 Scarpella, E., E.J. Simons, and A.H. Meijer. 2005. Multiple regulatory elements contribute to the vascular-spesific expression of the rice HD-Zip gene oshox1 in Arabidopsis. Plant Cell Physiology 46: 1400-1410. Shinozaki, K. and K.Yamaguchi-Shinozaki. 1997. Gene expression and signal transduction in water stress response. Plant Physiology 115: 327-334. Shinozaki, K. and K.Yamaguchi-Shinozaki. 2007. Gene networks involved in drought stress response and tolerance. Journal of Experimental Botany. 58: 221-227. Slamet-Loedin, I.H., J.L. Wibowo, S.Hutajulu, dan W. Rahayu. 1997. Penggunaan dua strain Agrobacterium tumefaciens super virulen untuk kokultivasi tanaman padi pada cv. cisadane dan rojolele. Prosiding Konggres I dan Seminar Ilmiah PBPI. Surabaya, 12-14 Maret 1997. Taylor, C.B. 1997. Comprehending cosuppression. The Plant Cell 9: 1245-1249 Wünn, J., A. Kloti, P.K. Burkhardt, G.C.G. Biswass, K. Launis, V.A. Iglesias, and I. Potrykus. 1996. Transgenic Indica rice breeding line IR-58 expressing a synthetic cryIA (b) gene from Bacillus thuringiensis provides effective insect pest control. Bio/Technology 14: 171-176. Yamaguchi-Shinozaki, K and K. Shinozaki. 2001. Improving plant drought, salt and freezing tolerance by gene transfer of a single stress-inducible transcription factor. in: Rice Biotechnology: Improving Yield, Stress Tolerance and Grain Quality – No. 236. (Novartis 1998. Transgenic Elite Indica Rice Varieties, Resistant to Xanthomonas oryzae pv. oryzae. Foundation Symposium). Chichester: Wiley. Zhang, J., R.J. Xu, M.C. Elliott, D.F. Chen. 1997. Agrobacteriummediated transformation of elite indica and japonica rice cultivars. Molecular Biotechnology 8: 223-231 Zhang, S., W.Y. Song, L. Chen, D. Ruan, N. Taylor, P. Ronald, R. Beachy and C. Fauquet. 1998. Transgenic elite indica varieties, resistant to Xanthomonas oryzae pv. oryzae. Molecular Breeding 4 (6): 551-558.

BIODIVERSITAS Volume 10, Number 2, April 2009 Pages: 70-75

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100203

Partial Characterization of a Novel Reovirus Isolated from a Hypovirulent Strain (9B21) of Cryphonectria parasitica (Murrill) Barr. SUPYANI

♥

Laboratory of Plant Protection, Faculty of Agriculture, Sebelas Maret University (UNS), Surakarta 57126. Received: 10th November 2008. Accepted: 26th December 2008.

ABSTRACT A filamentous fungus Cryphonectria parasitica (Murrill) Barr. is the causal agent of the chestnut blight disease. This fungus has some hypovirulent strains. These strains are known to be infected by viruses. To date, many species of viruses have been found to be associated with C. parasitica strains. The number genome segments, their relative size, titer and sequence homology as well as the effects on host fungal virulence and morphology were varied among the C. parasitica associating viruses. Cryphonectria parasitica mycoreovirus-1 (CpMYRV1) is a newly characterized reovirus isolated from C. parasitica. The viral genome consists of 11 segments of dsRNA termed S1 to S11. The aims of this research were to determine the complete sequences of the three shortest segments (S9-S11) of CpMYRV1, and to analyze the relationship of the dsRNA segments to the segments within the reoviruses genome. Sequence analyses showed that segments S9, S10, and S11 are 1072 bp, 975 bp, and 732 bp in size with single ORFs coding for 297, 247, and 101 amino acids respectively. Genome segments S9 to S11 have common terminal sequences, 5’GAUCA--- GCAGUCA3’. The deduced amino acid sequence coded by segment 9 showed a similarity to P11 of evolutionarily related Rosellinia anti-rot virus (Reoviridae), while S10 or S11 exhibited they were no significant sequence similarities to the known sequences published. © 2009 Biodiversitas, Journal of Biological Diversity Key words: Cryphonectria parasitica, CpMYRV1, molecular characterization.

INTRODUCTION The hypovirulence phenomenon on fungi caused by their association with viruses have been known since the discovery of swollen, superficial cankers and chestnut trees recovering from chestnut blight in Europe (Biraghi, 1953) and Michigan (Elliston et al., 1977; Day et al., 1977; Dodds, 1980). Many different types of dsRNA viruses have been found to be associated with filamentous fungus Cryphonectria parasitica (Murrill) Barr, the causal agent of the devastating chestnut blight disease. The number of dsRNA segments, their relative size, titer and sequence homology as well as the effects on host fungal virulence and morphology vary significantly among the dsRNAs examined (Dodds, 1980; Fulbright et al., 1983; Hiremath et al., 1986; Paul and Fulbright, 1988). At least four virus families (Reoviridae, Partitiviridae, Chrysoviridae and Hypoviridae) are known to infect C. parasitica and some confer hypovirulence (Hillman and Suzuki, 2004). Reoviruses are characterized by

 Correcponding address: Jl. Ir. Sutami 36 A, Kentingan Surakarta, 57126. Tel. & Fax.: +62-271-637457 e-mail: supyani@mail.uns.ac.id

distinct properties: (i) they have 10-12 dsRNA genomic segments mostly with a monocistronic nature; (ii) virus particles are multi-shelled; (iii) particles serve as viral mRNA synthesis factory. Cryphonectria parasitica mycoreovirus-1 (CpMYRV-1), Cryphonectria parasitica mycoreovirus2 (CpMYRV-2), and Rosellinia necatrix mycoreovirus3 (RnMYRV-3) are the members of family Reoviridae that infect fungi (Mertens et al., 2004). These viruses also have the ability to persistently attenuate virulence and stably alter complex biological processes upon infection of their fungal hosts (Enebak et al., 1994; Osaki et al., 2002; Wei et al., 2003). RnMYRV-3 is a newly characterized virus from Rosellinia necatrix Prillieux, an ascomyceteous fungus. This virus consisted of 12 segments dsRNA, ranging from ca. 0.41 bp to 2.95 kbp in size. All 12 dsRNA genomic segments of this virus have conserved terminal sequences at both 5’- and 3’ends. Double-shelled spherical particles approximately 80 nm in diameter are observed in a preparation from the mycelial tissue of the host. CpMYRV-1 and CpMYRV-2 are reoviruses recently isolated from C. parasitica. CpMYRV-2 was recovered from a canker in West Virginia in September 1989, whereas CpMYRV-1 was recovered in September 1982 from a virulent canker approximately 20 miles from where the CpMYRV-2

SUPYANI – Characterization of a mycoreovirus

was recovered, in the same district (MacDonald and Double, unpublished). Both viruses contain 11 segments of dsRNA. CpMYRV-2 contains 11 segments of dsRNA in equal molar amounts ranging from 1 to 5 kb in size, whereas isolate CpMYRV-1 is 0.7 to 5 kb (Enebak et al., 1994). The three largest dsRNAs of CpMYRV-1 were determined by Hillman et al. (2004). Genome segment 1 (S1) is 4127 bp in size with single ORF codes for 1354 amino acids. The size of S2segment was 3846 bp with single ORF codes for 1238 aa, whereas S3 was 3254 bp in size with single ORF codes for 1065 aa. S1 to S3 of the virus were found to have common terminal sequences (5’GAUCA --CGCAGUCA3’). Icosahedral particles with a doublelayered structure of approximately 80 nm in diameter were purified from the mycelial tissue of the host (Hillman et al., 2004). Regarding CpMYRV2, icosahedral particles approximately 60 nm in diameter were purified from the mycelial tissue of the host (Enebak et al., 1994). Only limited sequence data is available for this virus. The aims of this research were to determine complete sequences of the three shortest segments (S9-S11) of CpMYRV1, and to analyze the relationship of the dsRNA segments to the segments of other closely related reoviruses.

buffer in autoclaved cellophane tube for 2 hours at 200 volts. The samples then subjected to phenol chloroform extraction followed by ethanol precipitation, and the pellets were diluted in 20 L purified H2O. cDNA synthesis Double strands complementary DNA (cDNA) were synthesized as described by Gubler and Hoffman (1983) using a commercial kit (TimeSaver cDNA Synthesis Kit, Pharmacia). The procedure of cDNA synthesis is outlined schematically in Figure 1. Two micro grams dsRNA of each segment (of the three shortest segments) were used as templates. The product was subjected to phenol/chloroform extraction and purified using GeneClean II Kit (BIO 101) according to kit protocol. The cDNA was eluted in 15 L purified H2O. dsRNA dsRNA denaturation

Denatured dsRNA

MATERIALS AND METHODS Fungal strain and maintenance Strain 9B21 (hypovirulent, CpMYRV1 infected) of C. parasitica was a generous gift from Dr. William MacDonald (West Virginia University, Morgantown). A single conidial, virus-free isolate was obtained from the 9B21 strain as described by Hillman et al. (2004). Cultures were maintained on regeneration media at o 4 C in a refrigerator. CpMYRV1 dsRNA Extraction Extraction of CpMYRV1 dsRNA was done as described by Hillman et al. (1990) and Morris and Dodds (1979) with several modification. Fungal tissue (7.5 g) harvested from a 10-day-old culture of C. parasitica strain 9B21 in 300 mL potato dextrose broth was used as a material source. The dsRNA purification was performed using CC-41 cellulose according to the method of Morris and Dodds (1979). Double stranded RNA was analyzed by electrophoresis through 0.7 % agarose gel cast in TAE buffer. Gels were stained with ethidium bromide and photographed. CpMYRV1 dsRNA Segments Isolation For gene cloning purpose, each dsRNA segment was isolated. Total of 8, 2 g dsRNA (in 200 L) was run in 10% SDS-PAGE followed by slicing each band using a razor. The dsRNA segments were purified from the gel by electrophoresis using purification

First-strand cDNA synthesis

RNA:cDNA duplex

Second-strand cDNA synthesis

Double-stranded cDNA

GeneClean II kit

Ligated into SmaI-cleaved vector

Figure 1. The procedure of double-stranded cDNA synthesis.

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Ligation and transformation The blunt-ended cDNA were ligated into SmaIdigested pBluescript II SK (+/-) (Stratagene). Recombinant plasmids were transformed into competent E. coli strain DH5. The recombinant were grown on 500 L LB medium and spread on LB plate containing 50 ug/mL ampicillin, 50 g/mL 5-bromo-4chloro-3-indolyl-B-D-galactoside (X-gal, Takara) and 0.1 mM isopropyl-B-D- thiogalactopyranoside (IPTG, MBI). Selected recombinant plasmids were over night single cultured in LB containing 50 ug/mL ampicillin. Recombinant plasmids then purified from E. coli using QIAprep Spin Miniprep Kit (QIAGEN) according to the kit protocol. Sequencing and sequence analysis Sequencing reaction was prepared using Dye Terminator sequencing kit supplied by Applied Biosystems Division. Plasmids (250 ng) containing the inserted genes were subjected to PCR reaction containing primer and premix supplied by kit and amplified. Primers used in this reaction were general primers M13 F (for 3’ direction) and M13 R (for 5’ direction). PCR products were precipitated with ethanol and resolved in sequence dye and run on 50% polyacrylamide gel (super reading DNA sequence solution, Toyobo) diluted in TBE buffer with ABI Prism 377 Genetic Analyzer Sequence (Applied Biosystems). Sequence fragment were assembled TM with AutoAssembler 2.0 ABI Prism (Perkin Elmer) and analyzed using Genetic-Mac 10.0 (Software Development). Homology search was performed with the BLAST suite of programs from National Center for Biotechnology Information (NCBI). Terminal sequence determination To cover until both termini of each segment, a 5’rapid amplification of cDNA ends (5’-RACE) method as described by Polashock and Hillman (1994) and Hillman et al. (2004) was adopted. The cDNA segments were synthesized by RT reaction with virus specific primers (Table 1), tailed with oligo (dC), and amplified by PCR with nested virus-specific primers (Table 1), and abridged anchor primer. The PCR products were separated by agarose gel TM electrophoresis and purified by GeneElute Agarose Spin Columns (Sigma). The purified cDNA were ligated to pGEM®-T Easy Vector (Promega) and transformed to Competent High DH5 (Toyobo). The purified recombinant plasmids were sequenced according to the above method. Synthesis of full-length cDNA To construct a full-length cDNA clone of each segment, a set of primers pair from each terminal were designated and used in RT-PCR amplification. The full-length cDNAs were ligated to pGEM®-T Easy Vector (Promega), and transformed to Competent High DH5 (Toyobo).

RESULTS AND DISCUSSION CpMYRV1 dsRNA examination Several single conidial isolates derived from isolate 9B21 of C. parasitica were analyzed for the presence of dsRNA. Isolates that was similar in morphology to isolate 9B21 contained dsRNA, whereas those isolates that did not resemble isolate 9B21 were dsRNA-free. All 11 dsRNA segments found in isolate 9B21 were present in each of the dsRNA-containing single conidial isolates (Figure 2). Thus the dsRNA segments were transmitted to next generations in an all-or-none fashion. The genomic segments of CpMYRV1 were termed S1 to S11 with increasing order of mobilities in acrylamide gel as is for plant reoviruses. Order of migration of the segments was the same between agarose and acrylamide gels, although acrylamide gel gave better resolution. M

5 kb

1 kb

1 2 3 4 5 6 7 8 9 10

Figure 2. Polyacrylamide gel electrophoresis of the dsRNA extracted from mycelium of C. parasitica isolate 9B21. Segment 5 and 6 are similar in size and co-migrate on the gel. M, marker: 1 kb DNA Ladder.

Cloning of CpMYRV1 dsRNA The three largest of the 11 genomic dsRNA segments (S1-S3) of CpMYRV1 were sequenced earlier by Hillman et al. (2004). Thus, in this study the rest of smaller segments were targeted. From cDNA libraries derived from dsRNA of the three shortest segments, cDNA clones with relatively large inserts of over 400 bp were randomly chosen. By assembling sequences of cDNA clones (approximately 50 clones to each gel-isolated segments), three contigs were obtained. Three cDNA clones representing each of the 3 contigs were chosen, and used for the next analysis. Terminal structure The classic 5’RACE was used to determine the terminal sequence of S9 to S11, as used for S1 and

SUPYANI – Characterization of a mycoreovirus

S3 of CpMYRV1. Most 5’ RACE clones of plus strands of genome segments were 5’-GATCA----, while some lacked several most 5’ terminal nucleotides. The 5’ sequence, GATCA was confirmed by sequencing RLM-RACE clones (data not shown). The minus strands of genome segments were also determined with the classic RACE analysis to be 5’TGACTGC---. The 5’ pentamer, 5’GAUCA--- and 3’ octomer, ---CGCAGUCA3’ are conserved among the three largest segments of CpMYRV1 (Hillman et al., 2004). However, the eighth residue C from the 3 end was not shared in S9 and S11 as summarized in Table 1. As reported by Hillman et al. (2004), the 3’ end is similar to those of RnMYRV3 (---UGCAGAC3’) and coltiviruses (--a/uUGyAGUg/c3’), while 5’ terminus is similar to that of the genus Oryzavirus (5’GAU---) or coltiviruses (5’ GA---). Table 1. Primers couples used for 5’ RACE and their deoxyoligo-nucleotides. Segment 9

10 11

Primer name 9B9-3 9B95 9B91 9B90 9B9-4 9B10-3 9B10-4 9B10-2 9B10-1 9B83 9B82 9B100 9B101

Primer name and position 5’- direction () 3’- direction () 9B9-3 > 9B95 9B91 > 9B90 (172-189 nt) (90-107 nt) (751-769 nt) (807-826 nt) 9B90 > 9B9-4 (807-826 nt) (926-944 nt) 9B10-3 > > 9B10-4 9B10-2 > 9B10-1 (269-287 nt) (191-208 nt) (832-850 nt) (871-890 nt) 9B83 > > 9B82 9B100 > 9B101 (247-264 nt) (181-199 nt) (506-526 nt) (571-589 nt)

Deoxyoligonucleotides TCC CCA GCC CAA CCC GCA AGC GAT CGC CCC GCG CGA GCA

TGG ACT GAT CGC AAT GAA GTC AAT CTA CGA ACT TTA AGT

ACG ACG CAG ACG CCG TGA AAG GCC CAT GAA CAA CTG GCA

TGG GTG GAA ACC TTT CAC GGG CCC CCT ATT TTT CGC CCG

AAT AAA GCT TTT CTG CAA CGG TGC CAT GAG CTC CTG GCT

GAC GAC TGG CAC GAT CGC GTC GGA CAT TCG GGG ATG TTA

G CG C G C AG C G AG G

Sequences of CpMYRV1 S9-S11 The complete sequences of S9 to S11 were obtained from cDNA clones and RACE clones. As summarized in Table 2 the lengths of the CpMYRV1 genome segments range 1072 bp (S9) to 732 bp (S11), which are in accord with the agarose gel electrophoresis profile (Figure 2). S9 and S10 possess single large open reading frames (ORFs) spanning over 90% of their entire segment sizes. S11 is only an exception and has a relatively small ORF which corresponds to less than 50% of the entire segment sequence. Small ORFs comprising more than 300 nucleotides besides the large ORFs are

found on the minus strand of S9, while it remains to be answered whether those small ORFs are expressed. Table 2. Size of dsRNA segments S9, S10, and S11 of CpMYRV1 and their putative encoded proteins.

Segment S9 S10 S11

Total nucleotides (bp) 1072 975 732

Putative encoded proteins 32,880 kDa (297 aa) 27,758 kDa (247 aa) 11,461 kDa (101 aa)

As is the case with other reoviruses, each segment commonly consists of 5’ conserved pentanucleotide, (5’GAUCA--), and 3’ hepta-nucleotide (--GCAGUCA3’) conserved sequence (Table 3). Nucleotides at positions 6 to 8 from the 5’ are semiconserved (a/u-a/u-a/u). As reported for CpMYRV1 S1 to S3 (Hillman et al., 2004), no typical inverted repeat structure, which is found in plant reoviruses (Xu et al., 1989; Kudo et al., 1991), is formed by the terminal sequences adjacent to or the conserved terminal residues. The conserved terminal sequences and the number of genome segments are among very important criterion of reovirus taxonomy (Mertens et al., 2000). The tentative mycoreovirus genus is different from other genera in that the genus contains species with different numbers of genome segments: 11 for CpMYRV1 and CpMYRV2, and 12 segments for RnMYRV3. A hint to the problem of the genome segment number may come from the evolutionary scenario proposed by Hillman et al. (2004) that mycoreoviruses and coltiviruses might have diverged from a progenitor virus infecting an Acari arthropod species and having 12 genome segments. During the course of evolution the C. parasitica mycoreoviruses may have lost one of the segments which were dispensable for virus life cycle in the fungal host. Table 3. The 5’- and 3’-non-coding regions of dsRNA segments S9, S10, and S11 of CpMYRV1. Segment S9 S10 S11

5’ Non-coding region Size Terminal (bp) sequences 82 5’-GAUCAUUUGA… 74 5’-GAUCAAAAAT… 300 5’-GAUCAUAAUA…

3’ Non-coding region Terminal Size sequences (bp) …AUUGCAGUCA-3’ 91 …UACGCAGUCA-3’ 160 …AUUGCAGUCA-3’ 126

Interviral amino acid sequence similarities and their sequence motifs Osaki et al. (2002), Wei et al. (2003, 2004) and Hillman et al. (2004) noted the relatedness of four viruses: two fungus-infecting viruses, CpMYRV1 and RnMYRV3 and two members of the genus Coltivirus (CTFV and EyaV). The proteins encoded by the different dsRNA segments were designated PN, where N refers to the number of the RNA segment

B I O D I V E R S I T A S Vol. 10, No. 2, April 2009, pp. 70-75

CpMYRV1: 5 VVGSNIYDTTLLMTRKGQN-GAPDEVIPRPGFLTLLLNDIDSLRTRVELHNLIDNLN-LA 62 + +N T+++ R +N E+I +P FLTLL + E L NL+ RnMYRV3: 88 LTSNNFATATIVVERTNRNQNNRREIIAKPAFLTLLFSHKADALKNPEFRGL--NLDAFY 261

CpMYRV1: 63 TNEDYVKFAEYRTLFSQTTDMIRLAYTNGQPAVQTRATDSRTGS--VFYANTLTGDKAGN 120 + +D + E RT + + Y+ G P + R G V + L+G RnMYRV3: 262 STKDMMDTNEVRTRLGSGNGL-KFHYSKGVPTISQRGEAPVNGKMIVVWTPVLSGSSLHV 438

CpMYRV1: 121 LFRLLAPIAYRYLDVGLPRLFSYIHAQIGTTPAFRYNFDIQPIIKLAITNEPLDYGEWIG 180 L R++ + Y+ G+P +Y FD + N DY G RnMYRV3: 439 LNRVILSMYSEYVINGIPEFLAYAKTTYNAQTLNSVVFDSLTAARFIAMNVDFDYTNLFG 618

CpMYRV1: 181 ---QEG---------IHELERNVMIILSCSNITILAVLSIVGLGVGSHIMT 219 +G I E ERN ++ ++ S TI + GLG+ +H+M+ RnMYRV3: 619 VAPADGEPDPTDNLTIEEAERNYLLAVAPSQGTIAVAIQCGGLGL-AHLMS 768 Figure 3. The result of homology searching for putative protein encoded by segment 9 using BLAST program.

based on its electrophoretic mobility. Sequence database searches were performed with the newly determined sequences of CpMRYV1 P9 to P11 using the BLAST and FASTA programs. In addition to the previously identified equivalent segments, a BLAST search identified CpMRV1 S9/RnMRV3 S11. Sequence similarities (23% identity, Figure 3), and optimal scores among the four viruses were summarized in Table 4. CpMRV1 S10 or S11 did not yield any significant hit in BLAST or FASTA3 searches. It remains unclear which segments of RnMYRV3 and coltiviruses are counterparts of CpMYRV S10 and S11, which show no significant similarities to known sequences. Molecular properties of CpMYRV1 are characteristic of reoviruses and strengthen a suggestion made by Enebak et al. (1998), Osaki et al. (2001), and Hillman et al. (2004) that the CpMYRV1related mycoviruses make up a new genus within the family Reoviridae. These facts tempt to make a proposal to the International Committee of Virus Taxonomy (ICTV) that a new genus mycoreovirus is added to the family Reoviridae that contains CpMYRV1 as its type member, and two other members, C. parasitica mycoreovirus 2/C18 (CpMYRV2/C18) and R. necatrix mycoreovirus 3/W370 (RnMYRV3/W370). Recently, the proposal was approved, so the family Reoviridae now accommodates more than 11 genera with a large number of members infecting fungi, plant, invertebrates and vertebrates.

CONCLUSION A novel mycovirus (CpMYRV1) was isolated from a hypovirulent strain (9B21) of the chestnut blight fungus, Cryphonectria parasitica (Murrill) Barr. The CpMYRV1 genome consists of 11 segments of dsRNA termed S1 to S11. Sequence analyses showed that segments S9, S10, and S11 are 1072 bp, 975 bp, and 732 bp in size with single ORFs coding for 297 aa, 247 aa, and 101 aa, respectively. Genome segments S9 to S11 have common terminal sequences, 5’GAUCA --- GCAGUCA3’ which are identical to those of the previously sequenced segments. The deduced amino acid sequence coded by segment 9 showed a similarity to P11 of evolutionarily related Rosellinia anti-rot virus (Reoviridae), while S10 or S11 exhibited no significant sequence similarities to known sequences. These results support the previous suggestion that CpMYRV1 is a novel virus which belonging to the Reoviridae, acts as a hypovirulence factor on fungal host.

ACKNOWLEDGEMENTS The author is grateful to Dr. William MacDonald (West Virginia University, Morgantown) for the generous gift of Strain 9B21 (hypovirulent, CpMYRV1 infected) of C. parasitica.

SUPYANI – Characterization of a mycoreovirus

REFERENCES Biraghi, A. 1953. Possible active resistance to Endothia parasitica in Castanea sativa. Reports of the Congress of International Union of Forestry Research Organization. Rome 11: 643-645. Day, P.R., J.A. Dodds, J.E. Elliston, R.A. Jaynes, and S.L. Anagnostakis. 1977. Double-starnded RNA in Endothia parasitica. Phytopathology 67: 1393-1396. Dodds, J.A. 1980. Association of type 1 viral-like dsRNA with clubshaped particles in hypovirulent strains of Endothia parasitica. Virology 197: 1-12. Elliston, J.E., R.A. Jaynes, P.R. Day, and S.L. Anagnostakis. 1977. A native American hypovirulent strain of Endothia parasitica. Abstracts of Proceedings of American Phytopathological Society. 4: 83-84. Enebak, S.A., B.I. Hillman, and W.L. MacDonald. 1994. A hypovirulent Cryphonectria parasitica isolate with multiple, genetically unique dsRNA segments. Molecular Plant-Microbe Interactions 7 (5): 590-595. Fulbright, D.W., W.H. Weidlich, F.Z. Haufler, C.S. Thomas, and C.P. Paul. 1983. Chesnut blight and recovering American chestnut trees in Michigan. Canadian Journal of Botany 61: 3164-3171. Gubler, U. and B.J. Hoffman. 1983. A simple and very efficient method for generating cDNA libraries. Gene 25: 263-269. Hillman, B. I. and N. Suzuki. 2004. Viruses in the chestnut blight fungus. Advanced Virus Research 63: 423-72. Hillman, B.I., R. Shapira, and D.L. Nuss. 1990. Hypovirulenceassociated suppresion of host functions in Cryphonectria parasitica can be partially relieved by high light intensity. Phytopathology 80: 950-956. Hillman, B.I., S. Supyani, H. Kondo, and N. Suzuki. 2004. A Reovirus of the fungus Cryphonectria parasitica that is infectious as particles and related to the Coltivirus genus of animal pathogens. Journal of Virology 78 (2): 898-898. Hiremath, S.T., B. L’Hostis, S.A. Ghabiral, and R.E. Rhoads. 1986. Terminal structure of hypovirulent-associated dsRNAs in the chestnut blight fungus Endothia parasitica. Nucleic Acid Research 14: 9877-9896. Kudo, H., I. Uyeda, and E. Shikata, 1991. Viruses in the Phytoreovirus genus of the Reoviridae family have the same conserved terminal sequences. Journal of General Virology 72: 2857-2866.

Mertens, P. P. C., B. I. Hillman, and N. Suzuki. 2004. Genus mycoreovirus. In: Fauquet, C.M., M.A. Mayo., J. Maniloff., U. Desselbeger, and L.A. Ball. Virus Taxonomy: Eighth Report of the International Committee for the Taxonomy of Viruses. San Diego: Academic Press. Mertens, P.P.C., M. Arella, H. Attoui and 41 other authors. 2000. Reoviridae. In: Van Regenmortel, M.H.V., C.M. Fauquet, D.H.L. Bishop, E.B. Carstens, M.K. Estes, S.M. Lemon, J. Maniloff, M.A. mayo, D.J. McGeoch, C.R. Pringle, and R.B. Wickner. (ed.) Virus Taxonomy: Seventh Report of the International Committee for the Taxonomy of Viruses. New York: Academic Press. Morris, T.J., and J.A. Dodds. 1979. Isolation and analysis of double-stranded RNA from virus-infected plant and fungal tissue. Phytopathology 69: 854-858. Osaki, H., C.Z. Wei, M. Arakawa, T. Iwanami, K. Nomura, N. Matsumoto, and Y. Ohtsu. 2002. Nucleotide sequences of double-stranded segments from hypovirulent strain of he white root rot fungus Rosellinia necatrix: Possibly of the first member of the Reoviridae from fungus. Virus Genes 25 (1): 101-107. Paul, C.P., and D.W. Fulbright, 1988. Double-stranded RNA molecules from Michigan hypovirulent isolates of Endothia parasitica vary in size and sequence homology. Phytopathology 78: 751-755. Polashock, J.J. and B.I. Hillman, 1994. A small motochondrial double-stranded (ds) RNA element associated with a hypovirulent strain of the chestnut blight fungus and ancestrally related to yeast cytoplasmic T and W dsRNAs. Proceedings of National Academic Science USA. 91: 8680-8684. Wei, C. Z., H. Osaki, T. Iwanami, N. Matsumoto, and Y. Ohtsu. 2004. Complete nucleotide sequences of genome segments 1 and 3 of Rosellinia anti-rot virus in the family Reoviridae. Archives of Virology 149, 773-777. Wei, C. Z., H. Osaki, T. Iwanami, N. Matsumoto, Y. Ohtsu. 2003. Molecular characterization of dsRNA segments 2 and 5 and electron microscopy of a novel reovirus from hypovirulent isolate, W370, of the plant pathogen Rosellinia nectrix. Journal of General Virology 84: 2431-2437. Xu, Z., J.V. Anzola, and C.M. Nalin. 1989. The 3’-terminal sequence of a wound tumor virus transcript can influence conformational and functional properties associated with the 5’terminus. Virology 170: 511-522.

BIODIVERSITAS Volume 10, Number 2, April 2009 Pages: 76-80

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100204

Abundance and Diversity of Mould Inhabiting Muara Layang Estuary Sediment, Bangka Belitung Islands 1,♥

SEKAR LARASHATI , MUHAMMAD ILYAS

Research Centre for Limnology, Indonesian Institute of Sciences (LIPI), Cibinong-Bogor 16911. 2 Microbiology Division, Research Centre for Biology, Indonesian Institute of Sciences (LIPI), Cibinong-Bogor 16911. Received: 29th July 2008. Accepted: 16th October 2008.

ABSTRACT Estuary basin is a fertile area which has important role for sustaining many organisms from estuary and sea. Mould and other saprobic microorganisms have important role to decomposing organic material in estuary water. A study on diversity and abundance of mould inhabiting Muara Layang estuary sediment, Bangka Belitung Islands has not been conducted before. The objective of this study is to investigate the abundance and diversity of mould inhabiting Muara Layang estuary sediment, Bangka Belitung Islands. The mould isolation was based on sample dilution method with Rose Bengal Chloramphenicol Agar mould isolation media. The abundance of mould was counted by measuring the average Colony Forming Unit (CFU)/ml of all mould colonies which grown on isolation media by Total Plate Count (TPC) method. The diversity of isolated mould was identified based on phenotypic characters by observing both of macroscopic and 2 microscopic mould morphology. The result showed that the growth average of mould colonies is about (5-27.5) x10 CFU/mL. The result of mould identification showed that eight mould genera, Aspergillus (6 species), Chaetomium, Eupenicillium (3 species), Gliocladium, Paecilomyces, Penicillium (3 species), Scopulariopsis, Trichoderma (3 species), one group identified in class level (Coelomycetes), and three groups of unidentified sterile mould isolates were isolated. © 2009 Biodiversitas, Journal of Biological Diversity Key words: estuary, mould, sediment, abundance, diversity.

INTRODUCTION Estuary zone is an area with high nutrition and has an important role in transporting the nutrient into the sea (Mahasneh, 2001). Due to its fertility, estuary is used by fish and other aquatic biota as feeding, spawning and nursery ground. Microorganisms, such as bacteria and mould, play part in nutrient degradation and regeneration in estuarine. Degradation of leaves litter by microorganisms produce detritus which will be utilized through food chain by fish, shrimp, mollusk, benthic animal, and other aquatic biota (Chandramohan, 1984; Rao and Nair, 1986). Moulds have hypha with filament structures that can penetrate to the substrates. Their enzymatic ability is higher comparing to bacteria, especially in decomposing organic compounds like lignin and cellulose (Cromack and Caldwell, 1992; Mille-Lindblom, 2005). Moreover, moulds also produce extra cellular enzyme which can provide nitrogen source, vitamin and amino acid (Berg and

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Matzner, 1997). Varieties of moulds have been isolated from estuarine environment. Alternaria maritima, Aspergillus flavus, Aspergillus niger, Aureobasidium pullulans, Cladosporium sp., Humicola sp., Mucor sp., Penicillium sp., and Rhizopus sp. have been isolated from Korangi and Clifton bay, Pakistan (Mehdi and Saifullah, 1992). Acremonium alabamense, Aspergillus terreus, Penicillium crustosum, Penicillium purpurogenum, Phialophora fastigiata, Talaromyces flavus, and Trichoderma harzianum have been isolated from mangrove mud in Okinawa island (Ito and Nakagiri, 1997). Endophytic microfungi from Coelomycetes, Hyphomycetes, and Xylariales taxa are also isolated from vegetation in estuarine and mangrove area (Okane et al., 2001a, b). Nives-Rivera (2005) found number of moulds which are classified most in Ascomycetes dan Deuteromycetes taxa from estuarine in Puerto Rico. Muara Layang estuary in Bangka Belitung islands is an area which has low impact from tin mining, so it is planned to be the fishery conservation area. Research in physicochemical and biological have been conducted but the roles of microbes especially moulds in this area have not been conducted. Therefore, the aim of this research is to investigate the abundance and diversity of moulds inhabiting

LARASHATI & ILYAS – Mould inhabiting Muara Layang estuary sediment

Muara Layang estuary sediment in Bangka Belitung islands which is related to the physical and chemical factors in water of Muara Layang estuary.

MATERIALS AND METHODS Sampling location Sample of sediment was taken from Muara Layang estuary in Bangka Belitung islands in May 2007 (Table 1). Ekman dredge was used for taking sediment sample in each of sampling stations. Sediment was put in polybag or sterile bottle and either plated immediately or stored in a 4C refrigerator. Measurement of water quality such as pH, salinity, temperature, turbidity, and dissolved oxygen were done during the sampling by using Water Quality Checker DKK TOA (Table 2). Table 1. Description of sampling sites in Muara Layang Estuary, Bangka Belitung Islands Station

Sub area

Coordinate

Jelutung river estuary

Kelam river estuary

III

Lumut river estuary

Maras river estuary

Manjang river estuary

Melandur river estuary

S: 1°46’8.4” E:105°47’31.2” S: 1°47’15.72” E:105°48’42.12” S: 1°47’39.12” E:105°49’10.56” S: 1°48’25.92” E:105°48’44.64” S: 1°46’19.8” E:105°49’57” S: 1°46’53.16” E:105°52’11.64”

Sediment types Soft texture Soft texture Sandy texture Coarse and sandy texture Coarse and sandy texture Soft texture

Table 2. Data of physical and chemical quality in water of Muara Layang Estuary, Bangka Belitung Islands in May, 24, 2007 Station pH Turbidity Dissolved Oxygen Temperature Salinity NTU (mg/L) (°C) (‰) I 7.08 26.0 5.84 29.9 20.0 II 7.21 28.3 5.52 29.8 14.1 III 6.95 15.3 5.29 29.6 12.2 IV 6.99 16.2 5.76 30.3 12.2 V 6.85 26.6 5.27 29.4 7.9 VI 6.57 117.3 5.78 29.5 2.5

Isolation and enumeration of moulds Isolation was conducted in the laboratory with dilution method and the procedure will be done as followed (Ando et al., 2003; Ilyas et al., 2006): 10 g of sediment sample was weighed and put in test tube -1 with 90 mL sterile aquadest (10 dilution) then homogenized (preparation I). One mL of preparation I -2 was inoculated into 9 mL sterile aquadest (10

dilution) and homogenized (preparation II). The same -3 procedure was used to make 10 dilution (preparation III). 0.2 mL preparation I, II, and III was poured and spread on the Rose Bengal Chloramphenicol Agar. Each was plated in triplicate. Culture was incubated for 3-7 days in room temperature. Mould colony was counted in colony forming unit (CFU)/mL and chosen for isolation then transferred to Potato Dextrose Agar (PDA). Purification of mould colonies Mould cultures that grow during the isolation were purified with colony propagation by cutting and transferring aseptically part of mould mycelium to fresh culture media (Alexopoulos et al., 1996). The Isolates that grow on Rose Bengal was chosen and transferred to PDA chloramphenicol in a 6 cm diameter petri dish. Colonies was incubated for 3-7 days in 27ºC. The isolates were identified microscopically and macroscopically. The purified colonies were chosen and reculture in slant tube (PDA). The process was done in duplicate. Mould identification The isolated and purified moulds were identified base on phenotypic characters using guide books from Barnett (1955), Ellis (1971), Domsch et al. (1980), Sutton (1980), Webster (1980), Samson et al. (1995), Barnett and Hunter (1998), and Gandjar et al. (1999). Characteristic and morphology were identified both macroscopically and microscopically. The macroscopic characters consist of color and colony’s surface (granular, powder, elevate, smooth), texture, zonation, growing zone, radial and concentric line, reverse color, and exudates drop. Microscopic observation includes existence of hypha, hypha pigmentation, existence of clamp connection on hypha, form and ornamentation of spore and sporangiophore.

RESULT AND DISCUSSION The result demonstrated that the average of mould colonies from sediment sample of Layang estuary 2 was (5-27.5) x10 CFU/mL. The sequence of mould colonies number from the lowest to the highest was stated as follows. The lowest average of mould 2 colonies was 5.0 x10 CFU/mL obtained from station V in sub estuary of Manjang river. It was followed by station III in sub estuary of Lumut river which was 6.5 2 x10 CFU/mL. Station IV was in the third position in 2 terms of the average number which has 7.0 x10 CFU/mL, followed by station I and II in sub area of 2 Jelutung and Kelam river which has 18.5 x10 CFU/mL. The highest average of mould colonies 2 (27.5 x10 CFU/mL) was in station VI in sub area of Melandur river. Data of the abundance of mould colonies in each of the sampling site can be seen in Table 3.

B I O D I V E R S I T A S Vol. 10, No. 2, April 2009, pp. 76-80

Table 3. The average of mould colonies from sediment of Muara Layang Estuary, Bangka Belitung Islands, May 24, 2007 Station I II III IV V VI

Average of mould colonies 2 ( x10 CFU/ mL) 18.5 18.5 6.5 7.0 5.0 27.5

In general, the average of mould colonies in 6 sampling sites were low because they all have 4 average CFU ≤ 10 per gram or milliliter sample. Mould abundance was affected by environmental factors such as humidity, temperature, pH, nutrient and other chemical compound (Alexopoulos et al., 1996; Gandjar et al., 2006). One of the limiting factors for mould growth in 6 sampling sites was pH. Measurement of pH from 6 sampling sites showed that the pH tended to be neutral or base with its range about 6.57-7.21 (Table 2). Commonly, mould reaches its optimum growth in acid environment (Gandjar et al., 2006). Large numbers of the mould colonies have optimum growth in pH range between 5 and 6. Only a few can grow in a wide range of pH, for instances Aspergillus niger grows in pH between 2-8, Penicillium italicum grows in pH 1.9-9.3, and Fusarium oxysporum grows in pH 1.8-11.1 (Barnett and Hunter, 1998). Other factor that makes the abundance of mould colonies in 6 sampling sites was low in average CFU/mL was salinity. Osmotic pressure and (water activity/aw) will be affected by the salinity. As the salinity increases, the osmotic pressure will increase and water activity/aw will decrease. The low level of water activity has caused the mould difficult to take water from the environment for their metabolism activity. The result of salinity in 6 sampling sites showed that the salinity was between 2.5-20‰ (Table 3). According to Välikangas (1933), brackish water has salinity between 5-17‰ and sea water has 17‰ salinity. Generally, mould cannot be grown in high salinity. Only a few taxa can grow optimum in high salinity. Nives-Rivera (2005) found Chaetomium in hypersaline environment. Mould that can be grown in high salinity known as halophilic mould and can be isolated from mangrove, sea water, as well as food product such as salted fish (Gandjar et al., 2006). Even though halophilic mould that grows optimum in salinity between 5-20‰ is also found in brackish water, halophilic mould grows optimum in salinity between 25-40‰ (Rheinheimer, 1980). Mould is heterotrophic microorganism and generally grows as saprophyte. Organic compounds are needed as a nutrient source for their growth. The accumulation of organic compound in sediment tends to be higher compared to water body; therefore it can generate mould growth in sediment. Sediment texture

in 6 sampling sites was different. Based on the texture, station III, IV, and V have sand texture and station I, II, and VI have soft texture (Table 2). The average CFU showed that sediment texture also affected the number of mould colonies. The highest average of mould colonies was found in soft sediment texture. In general, sediment with soft texture contains higher organic matter than those that are in sandy texture. The presence of sufficient organic matter as a nutrition for mould growth will support the life sustainability of mould. Phenotypic identification showed that there are 23 different taxa which different morphologically (Table 4). The result of mould identification showed that eight mould genera, Aspergillus (6 species), Chaetomium, Eupenicillium (3 species), Gliocladium, Paecilomyces, Penicillium (3 species), Scopulariopsis, Trichoderma (3 species), whereas one group is identified in class level (Coelomycetes). Three groups of unidentified sterile mould isolates were isolated. They were sterile and did not form sexual reproduction structure during the incubation. Most of isolated mould colonies from 6 sampling sites were cosmopolite and saprophytic microorganisms. The most diverse of mould was from station I in sub area of Jelutung river. Station I had the highest variety in species, which was 12 different taxa. The isolates were Aspergillus spp.1, Aspergillus spp.3, Aspergillus spp.4, Eupenicillium sp.1, Eupenicillium sp.2, Gliocladium, Penicillium spp.2, Penicillium spp.3, Trichoderma spp.1, Trichoderma spp.2, and two groups of unidentified sterile mould. Isolation and identification result showed that mould from genera Aspergillus and Penicillium found in most of the sampling sites. Aspergillus and Penicillium are saprophytic mould that grows in variety of habitats (Samson et al., 1995). In estuarine area, these moulds live in sandy sediment, leaf litter and decay trunk (Nives-Rivera, 2005). Anand dan Sridhar (2004) reported that Penicillium and Aspergillus are terrestrial moulds which are commonly found around estuary. Numbers of mould isolating from estuary sediment such as Penicillium and Aspergillus were originally terrestrial mould. Their spores washed and diluted into river and then adapted and lived in estuarine (Jones and Hyde, 1988). Other saprophytic moulds which were found in sampling sites were mould from genera Chaetomium, Gliocladium, Paecilomyces, Scopulariopsis, and Trichoderma. Chaetomium was isolated from station II, Gliocladium was found in station I, V, dan VI, Paecilomyces was found in station III dan V, Scopulariopsis was found in station V, while Trichoderma was isolated from all of the sampling sites (Table 4). Chaetomium, Gliocladium, Paecilomyces, and Scopulariopsis can be found and isolated from estuarine environment (Nives-Rivera, 2005). While Trichoderma harzianum and Trichoderma sp. was isolated from rhizosphere and

LARASHATI & ILYAS – Mould inhabiting Muara Layang estuary sediment

Table 4. Mould generas and species that had been isolated from Muara Layang Estuary Sediment, Bangka Belitung Islands, May 24, 2007. Station I II III IV Aspergillus niger + Aspergillus spp.1, dark green colony + + Aspergillus spp.2, bluish green colony + + Aspergillus spp.3, brown colony + + Aspergillus spp.4, dark brown colony + + Aspergillus spp.5, brownish grey colony + Chaetomium sp. + Eupenicillium sp.1, brown colony + Eupenicillium sp.2, greenish yellow colony + + Eupenicillium sp.3, orange colony + Gliocladium sp. + Paecilomyces sp. + Penicillium spp.1, brown colony + Penicillium spp.2, grey colony + + Penicillium spp.3, greenish blue colony + + + + Scopulariopsis sp. Trichoderma harzianum Trichoderma sp.1, blackish green colony + + + Trichoderma sp.2, greenish yellow colony + + + Coelomycetes sp. + + Unidentified Sterile, cottony mycelia in yellowish white + + + + Unidentified Sterile, cotton mycelia in dark grayish + Unidentified Sterile, cottony mycellia in black + + Note: ( + ) = found, ( ─ ) = not found. Morphology Identification was done isolates that grow on PDA media, incubated in 27º C for 7-14 days. Generas

Species/explanation

mangrove mud (Ito and Nakagiri, 1997; Ito et al., 1999). Coelomycetes was isolated from station III and IV. These moulds known have strong association with plants. Coelomycetes is endophytic mould which has parasitic and mutualistic association with plants which become its host. That kind of relationship enables them to transfer their genetic matter (Tanaka et al., 1999). Coelomycetes is typically found in leaf litter. However, most of Coelomycetes was not sporulated while culturing in the laboratory (Itazaki et al., 1992). Number of those moulds can only be identified based on morphology reproduction structure in their host. Large numbers of Coelomycetes produce both sexual (teleomorph phase) and asexual (anamorph phase) spore in their natural habitat or hosts. Nevertheless, these moulds are sterile and tend to fail sporulating in culture media (Paulus et al., 2003). Some of Coelomycetes, such as Pestalotiopsis and Phoma, was isolated from mud and rhizosphere of mangrove plants (Ito et al., 1999).

CONCLUSION The average of mould colonies of Muara Layang 2 estuary sediment was between (5-27.5)x10 CFU/mL. The highest average abundance was in station VI in sub area of Melandur river. The result of mould identification showed that eight mould genera, Aspergillus (6 species), Chaetomium, Eupenicillium (3

V VI + + + + + + + + + + + + + + + + + + in mould

species), Gliocladium, Paecilomyces, Penicillium (3 species), Scopulariopsis, Trichoderma (3 species). In addition, one group was identified in class level (Coelomycetes), and three groups of unidentified sterile mould isolates were isolated. The highest diversity was found in station I in sub area of Jelutung river with 12 different species. Generally, result showed that the abundance of mould from 6 sampling sites was low and the diversity was dominated by cosmopolite and saprophytic moulds.

ACKNOWLEDGMENT

The authors would like to gratitude to Sulastri as the coordinator which supports this study and also to the team members for the assistance and cooperation. This study was supported by research program the development of conservation system co-management model and community empowerment of Layang estuary in Klabat bay, Bangka Belitung, Sub Program Competitive Project of East Kalimantan and Bangka Belitung, Indonesia Institute of Science 2006-2007.

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Domsch, K.H., W. Gams, and T.H. Anderson. 1980. Compendium of Soil Fungi. Vol. 1. London: Academic Press. Ellis, M.B. 1971. Dematiaceous hyphomycetes. London: Commonwealth Mycological Institute. Gandjar, I., R.A. Samson, K. van den Tweel-Vermeulen, A. Oetari, dan I. Santoso. 1999. Pengenalan Kapang Tropik Umum. Jakarta: Yayasan Obor Indonesia. Gandjar, I., W. Sjamsuridzal, dan A. Oetari. 2006. Mikologi Dasar dan Terapan. Jakarta: Yayasan Obor Indonesia. Ilyas, M., M. Rahmansyah, dan A. Kanti. 2006. Seri Panduan: Teknik Isolasi Fungi. Jakarta: LIPI-Press. Itazaki, H., K. Nagashima, Y. Kawamura, K. Matsumoto, H. Nakai, and Y. Terui. 1992. Cinatrins, a novel family of phospholipase A2 inhibitors. Taxonomy and fermentations of the producing culture: Isolation and structure of cinatrins. Journals of Antibiotics 45: 38-49. Ito, T and A. Nakagiri. 1997. Mycoflora of the rhizosphere of mangrove trees. IFO Research Communication 18: 40-44. Ito, T., I. Okane, and A. Nakagiri. 1999. Mycoflora of the rizhosphere of Salicornia europea L., a halophytic plant. IFO Research Communication 19: 34-40. Jones, E.B.G. and K.D. Hyde. 1988. Methods for the study of marine fungi from the mangroves. In: Agate, A.D., C.V. Subramanian and H. Vannuccie (eds.). Mangrove Microbiology. Role of Microorganisms in Nutrient Cycle of Mangrove Soils and Waters. New Delhi: UNDP/UNESCO. Mahasneh, A.M. 2001. Bacterial decomposition of Avicennia marina leaf litter from Al-Khor (Qatar-Arabian Gulf). Online Journal of Biological Sciences 1(8): 717-719. Mehdi, F.S. and S.M. Saifullah. 1992. Mangrove fungi of Karachi, Pakistan. Journal of Islamic Academic of Sciences 5 (1): 24-27. Mille-Lindblom,C. 2005. Interaction between Bacteria and Fungi in Aquatic Detritus: Causes and Consequences. [Dissertation]. Uppsala: Faculty of Science and Technology. Uppsala University.

Nieves-Rivera, A.M. 2005. Coastal Mycology of Puerto Rico: A Survey and Biological Aspects of Marine, Estuary, and Mangrove Fungi. [Dissertation]. Mayaguez, Puerto Rico: University of Puerto Rico. Okane, I., A. Nakagiri, and T. Ito. 2001a. Surculiseries rugispora gen. et sp. nov., a new endophytic mitosporic fungus from leaves of Bruguiera gymnorrhiza. Mycoscience. 42: 115-122. Okane, I., A. Nakagiri, and T. Ito. 2001b. Assemblages of endophytic fungi on Bruguiera gymnorrhiza in the Shiira river basin, Iriomoto island. IFO Research Communications 20: 4149. Paulus, B., P. Gadek, and K.D. Hyde. 2003. Estimation of microfungal diversity in tropical rainforest leaf litter using particle filtration: the effect of leaf storage and surface treatment. Mycological Research. 107(6): 748-756. Rao, T.S.S. and S.R.S. Nair. 1986. Tropic relationship in mangrove ecosystem. 2nd Introductory Training Course on Mangrove Ecosystems, Goa, India, November 1-25, 1984: Report of the Training Course. UNDP/UNESCO Reg. Proj. RAS/79/002. Rheinheimer, G. 1980. Aquatic Microbiology. London: John Wiley & Sons, Ltd. Samson, R.A., E.S. Hoekstra, J.C. Frisvad, and O. Filtenborg. 1995. Introduction to Food-Borne Fungi. Baarn, Holland: Centraalbureau voor Schimmelcultures. Sutton, B.C. 1980. The Coelomycetes. London: Commonwealth Mycological Institute. Tanaka, M. H. Sukiman, M. Takebayashi, K. Saito, M.S. Prana, and F. Tomita. 1999. Isolation, screening, and phylogenetic identification of endophytic plants in Hokkaido and Java Indonesia. Microbes and Environments 14 (4): 237-241. VĂ¤likangas, I. 1933. Ă&#x153;ber die biologie der ostsee als brackwassergebiet. Verhandlungen-Internationale Vereinigung fuer Theoretische und Angewandte Limnologie 6: 62-112. Webster, J. 1980. Introduction to Fungi. 2nd ed. Melbourne: Cambridge University Press.

BIODIVERSITAS Volume 10, Number 2, April 2009 Pages: 81-87

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100205

Population Status of Cacatua sulphurea parvula and Trichoglossus euteles in Alor, East Nusa Tenggara WAHYU WIDODO

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Zoology Division, Research Center for Biology, Indonesian Institute of Sciences (LIPI), Cibinong-Bogor 16911. Received: 11th November 2008. Accepted: 23rd January 2009.

ABSTRACT The aim of this survey was to know the abundance status of the Yellow-crested Small Cockatoo (Cacatua sulphurea parvula) and Yellow-headed Lorikeet (Trichoglossus euteles) in Alor, East Nusa Tenggara Province. There were four observation sites in the survey, namely: (i) Air Mancur preparatory Village (in and around of Tuti Adigae Natural Tourism Park, East Alor Sub-district), (ii) Tanglapui Village (Kampong Irawuri) (East Alor Sub-district), (iii) Probur Village (Kampong Wormanem) (Southwest Alor Sub-district), and (iv) Bota Village (Northwest Alor Sub-district). The survey was used for the bird encounter rates per unit time method. The bird population was estimated by abundance score value. Fifty species of birds or 64.9% of 77 species of birds occurring in Alor were recorded during field work. The yellow-headed lorikeet observed was more abundant with five abundance score and for the numbers per 10 hours observation were 58.06. There was no yellow-crested small cockatoo observed during the survey. However, the yellow-crested small cockatoo was presumed to be present in the fields by the direct observation of old nest site of Canarium trees. © 2009 Biodiversitas, Journal of Biological Diversity Key words: parrot, population, Cacatua sulphurea parvula, Trichoglossus euteles, Alor.

INTRODUCTION Psittacidae family is a special and unique group of parrots. Indonesia has less 76 species of parrots. Most of the parrots, namely 44 species are found in the middle of Papua forests, 20 species in Maluku; 16 species in Sulawesi; 14 species in Nusa Tenggara, five species in Kalimantan, four species in Sumatra and three species in Java, Bali and Madura (Sukmantoro et al., 2007). Among the species of parrots, Cacatua sulphurea parvula (yellow-crested small Cockatoo) and Trichoglossus euteles (yellowheaded Lorikeet) are found in the territory of Nusa Tenggara, especially in Alor. Because of the diversity of the body colors and smartness of such parrots in Indonesia attract fanciers, especially foreigner. This is shown by the increasing demand for export of such birds annually to several foreign countries, such as Europe, America or Asia, including Hong Kong, Singapore, Japan, and Saudi Arabia. In order to secure parrot export and keep their sustainability, the government c/q LIPI (the Biological Research Centre) as a scientific authority and Department of Forestry (PHKA) as the management

 Correcponding address: Jl. Raya Jakarta-Bogor Km 46, Cibinong 16911 Tel. & Fax.: +62-21-8765056 & 8765068 e-mail: wiedodo_318170@yahoo.com

authority must have cooperation annually to determine the quota of catch or export of such parrots proportionally. However, due to wide spread distribution of parrots in Indonesia, the determination of such quota is sometimes less likely supported by solid data or information on their population of parrots in their natural habitat. This caused by the fact that the survey taken on the population of such parrots mostly spreading the eastern part of Indonesia encountered many constrain, especially associated with the expensive cost of transportation. Nevertheless, LIPI (the Research Center for Biology) continuously conducting surveys in the past years on the population or abundance of Cacatua spp., parrot species in several Indonesian territories, namely in Buton (Adhikerana et al., 1997), Masa Lembo Isles (Darjono et al., 1997), Sumba (Hartini et al., 1997), East Flores (Hartini et al., 1998), North Halmahera (Widodo, 1998), Nusa Penida, Bali (Darjono and Hartini, 1999), South Tanimbar (Widodo, 1999), Lore Lindu National Park, Central Sulawesi (Widodo, 2001), and Manusela National Park, Seram (Widodo, 2006). In this paper, the survey findings on bird population or abundance in the territory of Alor, East Nusa Tenggara will be discussed. The specific objective of the survey is to obtain information on the population status of Cacatua sulphurea parvula and Trichoglossus euteles. In addition, this study also intended to record sources of their natural feed, resting

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trees and nesting places. This is very important to study so as the existence of such parrots as one of natural sources of economic value, will be well monitored their population and may be further utilized for conservation, not only for the benefit of Alor people, but also for the Indonesian nation in general. The data or information obtained from the survey findings may be expected to be used as advisement for its utilization.

MATERIALS AND METHODS The survey location was taken in Alor District, East Nusa Tenggara Province. There were four sites observation in the survey, namely: (i) Air Mancur preparatory Village (Tuti Adigae Natural Tourism Park, East Alor Sub-district); (ii) Tanglapui Village (Kampong Irawuri) (East Alor Sub-district); (iii) Probur Village (Kampong Wormanem) (Southwest Alor Subdistrict) and (iv) Bota Village (Northwest Alor Subdistrict). Vegetation uniquely grown at the survey area, Eucalyptus alba (paper bark tree), was especially found at the conservation area of Tuti Adigae Natural Tourism Park or “Taman Wisata Alam Tuti Adigae”. In addition, there were also Schleichera oleosa and Canarium commune. At the area bordering with the resident settlement around Tuti Adigae NTP area, Leucaena glauca, Cassia siamea, Ceiba pentandra, and Tamarindus indica were more dominant. At Irawuri site where it is bordered with Flores Sea, the paper bark tree was also found in abundance. The other unique plants were Casuarina equisetifolia and coast plant such as Terminalia catappa and the like. At Wormanem site, the vegetation seemed different with the two previous sites. Its vegetation consisted of forest of Aleurites moluccana and Canarium commune. In addition, Leucaena glauca was also introduced. Whereas the survey site at Bota area, most of observation area was taken at the beach side and the dominant plants are various species of mangrove. The weather condition, especially the rainfall more often takes place during the months of November up to February or March averagely of 1140 mm per year in Alor (Enga, 1991). The survey method to know the population status of Cacatua sulphurea parvula and Trichoglossus euteles found in Alor was the “bird encounter rates per unit time” (Bibby et al., 2000). This method was taken by recording total individuals of each species of parrots (Psittacidae) encountered in the surveys, but without measuring the distance of the observer to each individual bird encountered along the route or observation points. The aid employed in the observation of birds was the binocular of 8 x 30 mm. The survey findings recorded that total effective hours for the bird observation at all sites to be 31 hours.

For identification purpose of bird type species encountered during the surveys, a manual: “A Guide to the Birds of Wallacea Sulawesi, The Moluccas and Lesser Sunda Islands, Indonesia” written by Coates and Bishop (1997) was used. To know the feed source of parrots in natural habitat, the method of observation was used by “ad libitum” (Altman, 1974). This method was used conducted by observing a group of parrots doing feeding activity, then the species of trees and part of plants consumed were recorded. The bird activities in perching activities or resting at sleeping or nesting trees were also recorded. Furthermore, those plants were collected as vouchers, and made into herbarium to identify the scientific names of plants follow the classification system of Steenis et al. (2005). Data analysis on the population or abundance of parrots was taken by methods of totalling the individual birds at four sites and divided them into total observation hours, then the outcome obtained would be multiplied by 10 (Bibby et al., 1998). There were five scores employed to determine the abundance of birds, namely 1 or “rare” meaning the abundance score was less than 0.1; 2 or “uncommon” if its abundance score 0.1-2.0; 3 or “frequent”, if its abundance score 2.1-10.0; 4 or “common”, if its abundance score 10.1-40.0; and 5 or “abundant”, if its abundance score was more than 40.0.

RESULTS AND DISCUSSION Abundance score At the four survey sites, 50 species of birds (refer to Table 1) or 64.9% of all species of birds were found in Alor, where overall there were 77 species (White and Bruce, 1986). In Table 1, it appears that the bird species included in “abundant” under score five has just totaled one species (2%), whereas included in the “rare” category under score 1 was not found in the survey. Trichoglossus euteles is one of two species of parrots endemic in Nusa Tenggara under total individuals per 10 observation hours were 58.06. Its abundance score was classified as the highest, so that it may be stated that T. euteles was the most abundant. Even though, it spread area appears unevenly. The highest abundance of T. euteles was found in Tuti Adigae NTP site, situated at the Air Mancur preparatory Village. In each morning observation time taken at this site, the yellow-headed lorikeet could be encountered in group up to total 4050 birds. At Irawuri and Wormanem, the yellowheaded lorikeet was relatively less encountered just 1-2 birds. Even, the yellow-headed lorikeet could not be encountered at Bota mangrove forest. This site is not so far with the Bota settlements and its place developed into recreation areas. The abundance of the yellow-headed lorikeet in the area of Air Mancur is, among others, caused by the fact, that the area is

WIDODO – Cacatua sulphurea parvula and Trichoglossus euteles in Alor

fruits of fig Ficus racemosa and young fruits of Ceiba pentandra. The second highest abundance Total individual ∑ indi- Score Family Species birds at each sites vidual/ abunscore in the survey was four, namely I II III IV 10 jam dance for species of birds included under Phalacrocoracidae Phalacrocorax melanoleucos 0 1 0 0 0.32 2 “common”. Three species of bird Butorides striatus Ardeidae 0 0 0 1 0.32 2 under “common” category in the Elanus caeruleus Accipitridae 2 1 0 0 0.96 2 surveys were Dicrurus densus Haliastur indus 0 0 1 1 0.64 2 (12.90), Ducula aenea (12.58) and Accipiter fasciatus 4 0 0 0 1.29 2 Collocalia esculenta (10.64). Megapodius reinwardt Megapodiidae 0 1 2 0 0.96 2 Gallus varius Phasianidae 0 0 2 0 0.64 2 Whereas, there were 12 species of Actitis hypoleucos Scolopacidae 0 5 0 2 2.25 3 birds included in “frequent” category Numenius arquata 0 0 0 1 0.32 2 under score 3, namely: Philemon Numenius phaeopus 0 0 0 1 0.32 2 buceroides (8.38), Lonchura molucca Gallinago megala 0 0 0 6 1.93 2 (7.09), Nectarinia solaris (6.77), Sterna bergii Laridae 0 0 0 1 0.32 2 Zosterops citrinellus (6.45), Dicaeum Streptopelia chinensis Columbidae 4 8 4 0 5.16 3 Chalcophaps indica 4 7 0 0 3.55 3 igniferum (5.81), Streptopelia Geopelia maugei 6 3 0 0 2.90 3 chinensis (5.16), Chalcophaps indica Ptilinopus cinctus 0 2 1 0 0.96 2 (3.55), Geopelia maugei (2.90), Ducula aenea 13 9 17 0 12.58 4 Merops ornatus (2.90), Pachycephala Ducula rosacea 0 0 7 0 2.25 3 pectoralis (2.58), Actitis hypoleucos Macropygia magna 0 3 0 0 0.96 2 (2.25) and Ducula rosacea (2.25). Trichoglossus euteles Psittacidae 160 14 16 0 58.06 5 Eudynamys scolopaceus 2 0 1 0 Cuculidae 0.96 2 The rare category was not found, Caprimulgus affinis Caprimulgidae 4 0 0 0 1.29 2 but most (68%) of total species Collocalia esculenta Apodidae 6 13 14 0 10.64 4 observed were 34 bird species Halcyon chloris Alcedinidae 0 0 0 3 0.96 2 including “uncommon” category under Merops ornatus Meropidae 6 2 1 0 2.90 3 score two. The population was not Dendrocopos moluccensis Picidae 2 0 0 0 0.64 2 commonly found anymore, namely Hirundo tahitica Hirundinidae 0 0 10 2 0.64 2 Motacilla cinerea Motacillidae 1 2 0 0 0.96 2 total individual birds encountered by Campephagidae Coracina personata 1 0 0 0 0.32 2 every 10 observation hours was Dicrurus densus Dicruridae 23 13 4 0 12.9 4 between 0.1-2.0. Such condition Oriolus chinensis Oriolidae 0 3 2 0 1.61 2 should get seriously attention and Corvus macrorhynchos Corvidae 0 2 4 0 1.93 2 appropriately managed ecosystem. Parus major Paridae 0 2 1 0 0.96 2 The future anxiety is that the Saxicola caprata Turdidae 3 0 1 0 1.29 2 Gerygone sulphurea Acanthizidae 5 0 1 0 1.93 2 population status will change, to be Terpsiphone paradisi Monarchidae 2 3 0 0 1.61 2 reduced or rare under score 1 due to Hypothymis azurea 0 1 4 0 1.61 2 the various pressures of habitat Monarcha trivirgatus 1 0 0 0 0.32 2 quality and quantity. In Table 1, it is in Rhipidura rufifrons Rhipiduridae 2 0 1 0 0.96 2 detail based on the survey result on Pachycephalidae Pachycephala pectoralis 1 2 5 0 2.58 3 Artamus leucorhynchos Artamidae 0 0 3 0 0.96 2 the group of avis family (the bird Gracula religiosa Sturnidae 0 1 2 0 0.96 2 group) found under category Philemon buceroides Meliphagidae 10 13 2 1 8.38 3 “uncommon” with score 0.1-2.0 were Lichmera indistincta 3 0 1 0 1.29 2 Phalacrocoracidae, Ardeidae, Anthreptes malacensis Nectariniidae 2 0 0 0 0.64 2 Accipitridae, Megapodiidae, Nectarinia solaris 8 8 4 1 6.77 3 Phasianidae, Scolopacidae Dicaeum igniferum Dicaeidae 6 6 6 0 5.81 3 Zosterops citrinellus Zosteropidae 2 15 3 0 6.45 3 (Numenius arquata, Numenius Lonchura molucca Estrildidae 4 15 3 0 7.09 3 phaeopus, Gallinago megala), Lonchura quinticolor 0 5 0 0 1.61 2 Laridae, Columbidae (Ptilinopus Note: Scientific naming system of birds species follow Coates and Bishop cinctus, Macropygia magna), (1997). The survey sites: (i) Tuti Adigae NTP (Air Mancur); (ii) Irawuri Cuculidae, Caprimulgidae, (Tanglapui); (iii) Wormanem (Probur); (iv) Bota. Alcedinidae, Picidae, Hirundinidae, Motacillidae, Campephagidae, Oriolidae, Corvidae, Paridae, part of the conservation site of Tuti Adigae NTP, where its existence is relatively safer and protected. Turdidae, Acanthizidae, Monarchidae, Rhipiduridae, In addition, the condition of vegetation for its Artamidae, Sturnidae, Meliphagidae (Lichmera supporting feed such as trees of Schleichera oleosa indistincta, Anthreptes malacensis), and Estrildidae are flowering and its nectar is naturally the feed for (Lonchura quinticolor). the yellow-headed lorikeet in their natural habitat. At such site, the yellow-headed lorikeet is also fed on Table 1. Abundance score of species of birds in Alor during the survey.

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al. (1998) reported that C.s. parvula could be directly observed at Coordinate Size Population Sites References South East Lembata Isle at dense 2 (km ) status Latitude Longitude forest habitat, plantation o o Flores 8 30’ 121 00’ 13.540 14 Buchart et al. (1994) of Palmyra palm, o o Lomblen 8 25’ 123 30’ 329.7 Ttc Mochtar (1989) candlenut and canary-nut o o Adonara 8 20’ 123 10’ 497 Ttc Mochtar (1989) tree. The population of o o Solor 8 27’ 123 05’ 222 Ttc Mochtar (1989) C.s. parvula at Lembata o o Padar 8 40’ 119 35’ 20 ? PHPA/BirdLife Int’l (1998) o o Isle recorded was 25 Rinca 8 41’ 119 42’ 198 ? PHPA/BirdLife Int’l (1998) o o birds at the territory of Komodo 8 36’ 119 30’ 340 85-90 PHPA/BirdLife Int’l (1998) o o Nabatukan sub-district, Sumbawa 8 40’ 118 00’ 15.255 14 Setiawan (1996) nine birds at Labatukan Rare Johnstone et al. (1996) o o Moyo 8 12’ 117 32’ 349 Near threatened Johnstone et al. (1996) sub-district, respectively 10 Setiawan et al. (2000) two birds at o o Lombok 8 45’ 116 30’ 4.619 * Behrens (1995) Nagawuntung and o o Lembata/Larantuka 8 20’ 123 02’ 1.339 38 Hartini et al (1998) Wulangginting subo o Nusa Penida 8 44’ 115 32’ 202.84 6 Darjono and Hartini (1999) district. According to o o Alor 8 15’ 124 45’ 2.125 80 Setiawan et al (2000) o o Schmutz (1977), C.s. Pantar 8 25’ 124 07’ 712 29 Setiawan et al (2000) o o parvula was in Timor (West) 10 00’ 124 00’ 14.395 18 Setiawan et al. (2000) abundance in big group in Note: Ttc = not recorded the presence of C.s. parvula; ? = no new clues; *= already Flores in the 1970s. disappeared from Lombok. Whereas, C.s. parvula could still exist in Timor, though only several birds in 1990s (Noske, 1995). Distribution and population of Cacatua sulphurea Butchart et al. (1996) stated that the highest parvula population of C.s. parvula encountered in 1994 were The population of C.s. parvula in several sites that just 14 birds at Ria (western part of Flores). The cover the canary-nut tree forest area at Tuti Adigae NTP, Casuarina forest along Irawuri River, candle-nut population C.s. parvula in Sumbawa was stated to be plantation at Probur Village (Wormanem) and rare, even it is reported at Selah Legium to nearly mangrove forest at Bota, any such bird was not extinct, whereas at Moyo Isle included moderately encountered at the time of survey. However, several common, namely encountered in small group up to trees for their nesting places could still be found at the five birds at rain forest habitat and gardens at Brang bank of Mritang River, Kampong Wormanem, Probur Kua and Tanjung Pasir (Johnstone et al., 1996). Village (140-150 m above sea level). Nevertheless, some of the local hunters of birds said that the C.s. Benefit plants At least 26 species of plants are known to be parvula might still be encountered about seven birds in its habitat at the bank of Us River, at Kampong useful as source of feed, resting and nesting trees for Mahuwer, Mataraben Village. Based on the survey C.s. parvula and T. euteles in Alor (refer to Table 3). findings, it is estimated that though the C.s. parvula The T. euteles appears to more likely utilize nectar of may be found in Alor, it is likely to spread out toward blossoming flowers as source of their natural feed, the most eastern part of Alor Isle (Potomana among others, are Sesbania grandiflora, Mucuna mountainous forest), according to the report of local pruriens, Bombax valetonii, Ceiba pentandra, community where its forest condition is still good. Schleichera oleosa, Barringtonia racemosa and Setiawan et al. (2000) adds that the distribution and Nauclea orientalis. However, several kinds of fruits population of C.s. parvula in Alor was encountered also observed are fed by the yellow-headed lorikeet. along Kalabahi or Lendola River (six birds), Adigae The fruit plants are Eugenia javanica, Eugenia NTP (30 birds), along Taramana River (20 birds), jamboloides, Zizyphus jujuba and Mangifera indica. Takala (six birds), Pesomu (six birds), Halerman (four The observation findings indicated that the yellowbirds) and Tulen Dusun II (eight birds). It is further headed lorikeet is more commonly know as nectar reported that the C.s. parvula could be also found at feeder or feeding on fruits classified as soft fleshy Pantar under population of 29 birds, under total of 9 fruit. According to Monk et al. (1997), Trichoglossus birds spread over at Batuputih and 20 birds at type genus (T. haematodus, T. euteles, T. iris) Benggonang. According to Behrens (1995), C.s. includes “nectarivore”, where most of their sources of parvula could still be encountered in Alor and Pantar feed were nectar and flowery pollen in forest. in 1994, but the total number encountered was not Meanwhile, the yellow-crested small Cockatoo mentioned. Outside Alor, namely at Lomblen, appears to make use of fruits, flowers and seeds of Adonara and Solor in 1989, the population of C.s. plants, either of soft or hard fleshy fruits as their parvula was also surveyed, but there was no C.s. sources of feed. This is more likely in variety than that parvula recorded (Mochtar, 1989). However, Hartini et Table 2. Population status of C.s. parvula already surveyed in its several spread areas.

WIDODO – Cacatua sulphurea parvula and Trichoglossus euteles in Alor

Table 3. Benefit plants for C.s. parvula and T. euteles in Alor, East Nusa Tenggara.

trees. The height of holes for nests of such cacatua is situated at the height of 20 m above the ground Parrots level. The canary-nut tree stem of Benefit of plants for benefited Family name Species name *) parrots in Alor 117 cm in diameter, by the local from hunters, was set up pin made of SbP PhB PhT C.s. p T. e bamboo to climb up and pick up Andropogon sorghum** Gramineae +(bj) √ young cacatua at breeding season. Borassus flabellifer Palmae +(bh) √ According to the local hunters, Cocos nucifera Palmae +(bt) √ Amorphophallus sp.*** Araceae +(bh)+(bj) √ usually in December the Cacatua Musa paradisiaca Musaceae +(bh) √ √ pair will use such nesting hole to lay Casuarinaceae Casuarina equisetifolia +(bt) √ eggs and raise young cacatua after Ficus benjamina** Moraceae +(bh) +(bt) √ incubating and hatching periods. Artocarpus communis Moraceae +(bh) √ In one egg laying period, it was Artocarpus integra Moraceae +(bh) √ reported that cacatua would lay two Moringa oleifera** Moringaceae +(bh)+(ne) √ √ eggs. In January or February, the Caesalpiniaceae Bauhinia tomentosa +(bt) √ hunter would harvest such young Caesalpiniaceae Tamarindus indica** +(bh) +(bt) √ cacatua because it was estimated Papilionaceae Erythrina variegata +(ne) √ Papilionaceae Erythrina fusca** +(bu) +(bt) √ in such months the time had arrived Papilionaceae Sesbania grandiflora*** +(bh)+(ne) √ √ for young cacatua to be able to fly. Papilionaceae Mucuna pruriens*** +(ne) √ According to local bird hunters, Canarium oleosum*** Burseraceae +(bh) √ Cacatua sulphurea parvula were Canarium commune Burseraceae +(bh) +(bt) √ encountered its nest on canary-nut Garuga floribunda** Burseraceae +(bt) √ tree grown at Kampong Likuatang Euphorbiaceae Aleurites moluccana** +(bh) √ (West Lembur), and at Kampong Anacardiaceae Mangifera indica +(bh) √ √ Elang (Kokar). If not on canary-nut Anacardiaceae Spondias pinnata +(bh) √ trees, the C.s. parvula would make Schleichera oleosa Sapindaceae +(ne) √ Zizyphus jujuba Rhamnaceae +(bh) √ nest on stem of paper bark tree, Bombacaceae Bombax valetonii +(ne) √ many of which were previously Bombacaceae Ceiba pentandra +(ne) +(bt) √ √ found at Lembur. One nesting hole Sterculia foetida** Sterculiaceae +(bt) √ would be occupied by a pair of Pterocymbium javanicum Sterculiaceae +(bt) √ cacatua and its two young. At the Carica papaya*** Caricaceae +(bj) √ time of entrapping, the cacatua and Sonneratiaceae Duabanga moluccana** +(bt) √ its young would be all harvested Lecythidaceae Barringtonia racemosa +(ne) √ from the nesting hole, so that the Eucalyptus alba Myrtaceae +(bt) +(bt) √ √ harvest system as such had Eugenia javanica Myrtaceae +(bh) √ Eugenia jamboloides Myrtaceae +(bh) √ threatened the conservation of Planchonia valida*** Sapotaceae +(bt) √ cacatua in Alor, and in the territory Alstonia scholaris*** Apocynaceae +(bt) √ of Nusa Tenggara in general. Nauclea orientalis Rubiaceae +(ne) √ In several references are also Asclepiadaceae Calotropis gigantea +(bh)+(bj) √ stated that the type of tree stem Tetrameles nudiflora ** Datiscaceae +(bt) √ known as nesting place for the C.s. Note: *) Classification system of scientific names of plants follow Steenis et al. parvula are Cocos nucifera, Ficus (2005); **) Source is from Setiawan (1996). ***) Hartini et al. (1998). SbP = benjamina, Tamarindus indica, Source of feed; PhB = nesting trees; PhT = sleeping/perching trees; C.s.p = Ceiba pentandra, Pterocymbium Cacatua sulphurea parvula, T.e = Trichoglossus euteles. Bh = fruit, bj = seed, bt javanicum, Planchonia valida and = stem, ne = nectar. Alstonia scholaris. This indicates that the nesting type for the yellowcrested small cockatoo would be in the statement of Monk et al. (1997), who has the hard stem and soft stem as well. However, C.s. classified Cacatua sulphurea as “granivore-arboreal”. parvula were reported to nest generally on canary Several sources of references state that the tree. nesting trees of Cacatua sulphurea in Nusa Penida Different from the C.s. parvula, the T. euteles Isle (Bali) were at Sterculia foetida and “kutuh” trees found at Kampong Wormanem, nest on Eucalyptus at the height of nesting hole above 6-10 m of ground alba (paper bark tree) at the height of about 4 m level (Setiawan et al., 1996). Cacatua sulphurea was above ground surface and about 80 cm deep. also nesting on Tetrameles nudiflora in Sumbawa at However, the nesting trees of T. euteles on the the height of nesting hole above 10 m of ground level. Eucalyptus alba also often used in turn by Gracula Nonetheless, based on the survey observation religiosa. However, Gracula religiosa also nest on findings over their used nests, it appears that cacatua candlenut and canary-nut trees. in Alor seems to choose holes on still live canary-nut

B I O D I V E R S I T A S Vol. 10, No. 2, April 2009, pp. 81-87

Trading aspect and their conservation The trading of C.s. parvula is estimated to have last for sufficiently long time, though its large-scale exploitation of forest was estimated to take place in the 1980s (Setiawan et al., 2000). To anticipate the increasing possibility of C.s. parvula all gone from nature, such species should be also stated as globally threatened-extinct (Shannaz et al., 1995). Nevertheless, the trading of both species of parrots from Alor sometimes still maintains/exist. This case was monitored from the survey findings at several bird markets such as in Jakarta 31 birds were sold (Widodo, 2002). At several bird markets in Semarang and Surabaya, C.s. parvula were monitored to be two and five birds sold at Rp 300,000 up to Rp 400,000 per bird The period of 1997-2001, the trading of C. sulphurea in Medan, 400 birds were recorded (Shepherd et al., 2004). The luster of trading of C. sulphurea in Medan seems to be induced by demand for meeting the needs of local markets as well as traders from Singapore and Malaysia. This condition is different from the trading of T. euteles, which appears to be relatively less in number, namely monitored just five birds at exporter level in Jakarta within the period of April 1999 up to December 2000, and in Medan 11 birds within the period of 1997-2001 (Shepherd et al., 2004). Viewed from the catch quota of the yellow-crested small Cockatoo from year in year out has been likely decreased in number (refer to Table 4). Table 4. Total catch quota of the yellow-crested small cockatoo for export by 1984-1991*). Years

C.s. parvula (NTT)

C.s. parvula (NTB) ∑ (birds)

1984 2250 1985 1500 1986 1987 550 1988 550 1989 725 1990 1100 1991 2950 Total 9625 quota Note: *) PHPA/BirdLife-IP (1998).

625 500 250 250 300 265 2190

2875 2000 800 800 1025 1365 2950 11815

The decrease in total catch quota of the yellowcrested small cockatoo is foreseen as relating to the increasing difficulty of such commodities entrapped from its habitat. This is supported by the fact that based on the monitoring on population study of the yellow-crested small cockatoo in its habitat in the 1995s was began to be rare, even one bird could not be encountered in its previous habitat. According to Trainor (2002), that the biggest threat against the habitat and the species in Nusa Tenggara was illegal logging, forest fire, expansion of plantation cultivation area, considerable chopping down of grazing and hunting. It should be immediately followed with

concrete step by the government, that the disappearance of the habitat and forest degradation in Nusa Tenggara and the impact of bird utilization accompanied by the uncontrolled catches that has caused several types of such natural resource extinct could be avoided. On the other side, Cacatua sulphurea has been included in the protected species based on the Decree of the Minister of Forestry of Republic of Indonesia as spelt out under No. 350/Kpts-II/1997 and No. 522/Kpts-II/1997 on the utilization of wild flora and fauna. The yellow-headed Lorikeet is included under protected species. This is verified further under Government Regulations No 7 of 1999 on the Conservation of Species of Plants and Fauna. Fauna has been categorized as extinctthreatened species should there have been any special treatment with tight rules. In article 11, paragraph one of Government Regulation of the Republic of Indonesia No. 8 of 1999, it is elucidated that the breeding outcome of any wild animal that may be used for commercial purpose shall be the second and it’s following generations.

CONCLUSION The survey findings of two species of parrots found in Alor appear that just the yellow-headed Lorikeet (Trichoglossus euteles) directly encountered fewer than five abundance score and total individual per 10 observation hours has been 58.06. Whereas, the yellow-crested small Cockatoo (Cacatua sulphurea parvula) could be directly encountered, but identified from the trees used for its nesting place, namely on canary-nut trees at the bank of Mritang River, Kampong Wormanem, Probur Village,. The recommendation for cultivating the population of the Cacatua sulphurea parvula should enjoy a robust priority in West Nusa Tenggara and East Nusa Tenggara. The restoration of species should be immediately followed up, as its current population has been very small in number.

REFERENCES Adhikerana, A.S., A. Saim, M. Muslihun dan M. Amir. 1997. Status Burung Paruh Bangkok di Pulau Buton. [Laporan Teknik Proyek Litbang dan Pendayagunaan Biota Darat Tahun 1996/1997]. Bogor: Puslitbang Biologi-LIPI. Altman, J. 1974. Observational study of behaviour: sampling methods. Behaviour 49: 227-265. Behrens, S. 1995. Ein hilfruf fűr den gelbwangenkakadu Cacatua sulphurea. Papageien 8: 280-285. Bibby, C.J , M. Jones and S. Marsden. 1998. Expedition Field Techniques, Bird Surveys. London: Royal Geographical Society. Bibby, C.J., N.D. Burgess, D.A. Hill and S. Mustoe. 2000. Bird Census Techniques. 2nd ed. London: Academic Press. Buchart, S.H.M., T.M. Brooks, C.W.N. Davies, G. Dharmaputra, G.C.L. Dutson, J.C. Lowen, and A. Sahu. 1996. The conservation status of forest birds on Flores and Sumbawa, Indonesia. Bird Conservation International 6: 335-370.

WIDODO – Cacatua sulphurea parvula and Trichoglossus euteles in Alor

Coates, B.J. and K.D. Bishop. 1997. A Guide to the Birds of Wallacea Sulawesi, The Moluccas and Lesser Sunda Islands, Indonesia. Alderley: Dove Publications. Darjono dan S. Hartini. 1999. Survei Burung Kakatua-putih jambulkuning (Cacatua sulphurea) di Nusa Penida, Bali. [Laporan Teknik Proyek Litbang dan Pendayagunaan Biota Darat Tahun 1998/1999]. Bogor: Puslitbang Biologi-LIPI. Darjono, S. Hartini, Suparno, A. Sujadi dan I. Setiawan. 1997. Survey Burung Cacatua sulphurea abboti (Psittacidae) di Pulau Masakambing, Kep. Masalembo, Kab. Sumenep, Prop. Jawa Timur. [Laporan Teknik Proyek Litbang dan Pendayagunaan Biota Darat Tahun 1996/1997]. Bogor: Puslitbang Biologi-LIPI. Enga, A.H. 1991. Welcome to the Island of Moko drums, A Guide Book for Touring in Kabupaten Alor. Kalabahi: Bapparda TK II Alor. Hartini, S., D. Astuti, Suparno dan M. Amir. 1998. Burung Paruh Bengkok di Kabupaten Flores Timur, Nusa Tenggara Timur. [Laporan Teknik Litbang Model dan Teknik Penangkaran Satwa Langka (Burung Paruh Bengkok) Tahun 1997/1998]. Bogor: Puslitbang Biologi-LIPI. Hartini, S., Darjono, W. Widodo, Suparno, M. Amir dan I. Setiawan. 1997. Burung-burung Paruh Bengkok (Psittacidae) di Pulau Sumba, Nusa Tenggara Timur. [Laporan Teknik Proyek Litbang dan Pendayagunaan Biota Darat Tahun 1996/1997]. Bogor: Puslitbang Biologi-LIPI. Johnstone, R.E., P. Jepson, S.H.M. Butchart, J.C. Lowen and D. Prawiradilaga. 1996. The birds of Sumbawa, Moyo and Sangeang Islands, Nusa Tenggara, Indonesia. Records of the Western Australian Museum 18: 157-178. Mochtar, D. 1989. Laporan Inventarisasi Satwa Liar Kakatua Jambul Kuning (Cacatua sulphurea) dan Beo (Gracula religiosa) di Kabupaten Flores Timur, Kupang: Perlindungan Hutan dan Pelestarian Alam. Monk, K.A., J. de Fretes, and G. Reksodiharjo-Lilley. 1997. The Ecology of Nusa Tenggara and Maluku. Singapore: Periplus Editions (HK) Ltd. Noske, R.A. 1995. At the crossroads of two avifaunas-Timor. Oriental Bird Club Bulletin 21: 34-38. PHPA/BirdLife-IP, 1998. Rencana Pemulihan Kakatua-kecil Jambul-kuning. Bogor: PHPA/BirdLife International-Indonesia Programme. Schmutz, E. 1977. Die vögel der Manggarai (Flores), 2. Niederschrift. Privately published, Ruteng, Flores.

Setiawan, I. 1996. Status Cacatua sulphurea di Pulau Nusa Penida, Bali dan Pulau Sumbawa, Nusa Tenggara Barat. Bogor: PHPA/BirdLife International-Indonesia Programme (Laporan no. 6). Setiawan, I., A. Jati, D. Lesmana, C. Trainor dan D. Agista. 2000. Telaah Awal Status dan Penyebaran Kakatua-kecil Jambulkuning (Cacatua sulphurea parvula) di Pulau Alor, Pantar, Timor Barat, Flores dan Moyo. Bogor: PKA/BirdLife International-Indonesia Programme (Laporan No. 12). Shannaz, J. P. Jepson dan Rudyanto. 1995. Burung-burung Terancam punah di Indonesia. Bogor: PHPA/BirdLife International-Indonesia Programme. Shepherd, C.R., J. Sukumaran and S.A. Wich. 2004. Open Season: An Analysis of the Pet Trade in Medan, Sumatra 1997-2001. Petaling Jaya, Selangor: Traffic Southeast Asia,. Steenis, C.G.G.J van. 2005. Flora untuk Sekolah di Indonesia. Jakarta: PT Pradnya Paramita. Sukmantoro, W., M. Irham, W. Nuvarino, E. Hasudungan, N. Kemp and M. Muchtar. 2007. Daftar Burung Indonesia no.2. Bogor: Indonesia Ornithologist’s Union. Trainor, C.R. 2002. Birds of Gunung Tambora, Sumbawa, Indonesia: effects of altitude, the 1815 cataclysmic vulcanic eruption and trade. Forktail 18: 49-61. White, C.M.N. and M.D. Bruce. 1986. The Birds of Wallacea (Sulawesi, The Moluccan and Lesser Sunda Island, Indonesia). An Annotated Checklist. BOU. Checklist No.7. Bogor: British Ornithologist’s Union. Widodo, W. 1998. Kelimpahan, habitat dan pakan alami burungburung paruh bengkok (Psittacidae) di Halamhera Utara. Ekologi Indonesia 2 (3): 12-18. Widodo, W. 1999. Kelimpahan dan pakan alami burung-burung paruh bengkok (Psittacidae) di Tanimbar Selatan. Gakuryoku 5 (3): 169-175. Widodo, W. 2001. Studi populasi burung-burung paruh bengkok (Psittacidae) di Taman Nasional Lore Lindu, Sulawesi Tengah. Gakuryoku 7 (1): 75-80. Widodo, W. 2002. Perdagangan Burung Paruh Bengkok (suku Psittacidae) di Jabotabek. Seminar Nasional IX PERSADA. IPB Baranangsiang, Bogor, 19 Maret 2002. Widodo, W. 2006. Kemelimpahan dan sumber pakan burungburung di Taman Nasional Manusela, Seram, Maluku Tengah. Biodiversitas 7(1): 54-58.

BIODIVERSITAS Volume 10, Number 2, April 2009 Pages: 88-93

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100206

Reef Fish Status in Northern Acehnese Reef Based on Management Type 1,♥

EDI RUDI , SAYYID AFDHAL ELRAHIMI , TASRIF KARTAWIJAYA , YUDI HERDIANA , 2 2 2 3 FAKHRIZAL SETIAWAN , SHINTA T. PARDEDE , STUART J. CAMPBELL , JERKER TAMELANDER 1

Faculty of Science, Syiah Kuala University (UNSYIAH), Banda Aceh 23111 2 Wildlife Conservation Society (WCS) Indonesian Program, Bogor 16151 3 IUCN, Coastal Oceans Research and Development-Indian Ocean (IUCN-CORDIO), Rue Mauverney 28, CH 1196

Received: 12th December 2008. Accepted: 23rd February 2008.

ABSTRACT The objectives of this study were to provide reliable data and information on reef fish in the northern Aceh region of Weh and Aceh islands based on management types. This research use underwater visual census techniques at 29 sites around Weh Island and Aceh Islands. Reef fish abundance in area that were protected under the management of Panglima Laot of -1 -1 Sabang Weh Island (32505 no.ha ) and biomass (748 kg.ha ) were also significantly higher than in Aceh Islands which is -1 -1 open access areas (abundance=9539 no.ha ; biomass=396 kg.ha ). Fish species from the family Pomacentridae (damsel fish) were highest in abundance and biomass at northern Acehnese reef. Population of carnivorous fish such as parrotfish (Family Scaridae) was low in all study sites which indicated that northern Acehnese reef is lack of reef resilience both in managed and open access area. Higher numbers and abundance of reef fish in marine managed areas, such as Panglima Laot in Sabang, could be considered for further conservation purpose of marine resources. © 2009 Biodiversitas, Journal of Biological Diversity Key words: Coral reef, reef fish, management type, northern Acehnese reef

INTRODUCTION The reefs at northern Acehnese are dominated by massive species (mainly Poritidae and Faviidae) intermingled by patches of branching Acropora and Montipora species in sheltered areas and mostly branching species at high-energy reefs (Brown, 2005). Weh Island and Aceh Islands are the two main islands in northern Aceh and also the western most reef areas in the Indonesian archipelago. The marine fauna and flora of north-west Sumatra, including the northern Aceh reefs are comprised of species from the Indian Ocean and the Pacific Ocean and make the region biogeographically distinct from eastern Indonesian coral reef species. Coral reefs of Weh Island are known to be generally in better condition than those on Aceh islands because of past differences in management between these two regions of northern Aceh. The reefs are important for fisheries with a range of artisenal gears used and mainly pelagic commercial fisheries. The area is known for its tourism industry  Correspnding address: Kampus Unsyiah, Darussalam, Banda Aceh 23111 Tel. & Fax.: +62-651-7428212; +62-651-7410248 e-mail: edirudi@yahoo.com

with snorkeling and SCUBA diving and other recreational activities as the main attractions. These activities contribute income to coastal communities, in addition to farming, business, and government sectors (Baird et al., 2005). Coral reefs habitats and their associated fauna and flora are therefore important marine ecosystems with important ecological functions and services in the region. Although these reefs were subject to substantial disturbance from the tsunami, initial claims that northern Aceh reefs were destroyed or greatly impacted by the tsunami in 2004 were unfounded (Brown, 2005). Nevertheless damage to coastal communities was severe with five of thirteen villages in Weh Island heavily affected by the earthquake and tsunami; mostly the northern and southern coasts. Damaged houses, boats, fishing gears, and loss of life paralyzed the economic state of northern Aceh’s fisheries, including the destructive fishing activities. However, overfishing and destructive fishing practices had caused serious damage and significantly degraded coral reef ecosystems in Aceh Islands prior to the tsunami (Baird et al., 2005). The reefs of Northern Aceh range from those are very poor to those in good condition but generally have only been recently well documented (Baird et al., 2005; Campbell et al., 2005a,b; Rudi, 2005; Ardiwijaya et al., 2007). To improve the management

RUDI et al. â&#x20AC;&#x201C; Reef fish status in Northern Acehnese

of these reefs and prevent damaging, unsustainable, and illegal fishing practices from returning, investment is required to implement management practices and strategies that help rehabilitate, restore and protect marine resources through programs of regular monitoring, education, coastal management and establishment and maintenance of marine protected areas. Wantiez et al. (1997) and Aswani et al. (2007) concluded that species richness, density and biomass of fish on the protected reefs were higher and statistically significant compare with open access reef. Indonesia possesses the worldâ&#x20AC;&#x2122;s richest reef fish fauna. Stretching in an east-west direction for approximately 5,000 km and embracing nearly 18,000 islands, the archipelago features a seemingly endless array of marine habitats. Although universally acclaimed as the leading reef fish locality, there is no comprehensive, published faunal list to substantiate this assertion. Randall (1998) proposed several key factors for the proliferation of coral reef species in the East Indian region, i.e. Weh Island and surrounding areas. Study of Allen and Adrim (2003) indicates that 6 species of reef fishes are currently considered Acehnese endemics. The aim of this research was to provide reliable data and information on coral reef resources of northern Aceh especially reef fish based on management type. Hopeful, the results would be useful for local people, scientists, tourists, and other stakeholders to evaluate the reef fish status and be used to evaluate the effectiveness of future marine resources management.

MATERIAL AND METHODS Coral reef surveys was conducted at 21 sites; consisting of 13 sites on Weh Island and 8 sites on Aceh Islands. Coral reef fish surveys were conducted at 29 sites; consisting of 19 sites on Weh Island and 10 sites on Aceh Islands (Figure 1). Sites were selected to represent the reefs of the region and the types of site management including: Marine Protected Area (MPA, consisted of Panglima Laot and Tourism Reserve) where fishing restrictions are in place and unrestricted fishing or open access areas. The method used in this research was underwater visual census technique (VCT) for reef fishes (English et al., 1997; Hill and Wilkinson, 2004). In order to obtain representative data of the reef, transects were laid at two waters depths at 2-3 m (shallow) and 6-8 m (deep). Data were collected at the species level and 9 size classes visually estimated (0 cm, 5-10cm, 10-15 cm, 15-20 cm, 20-25 cm, 25-30 cm, 30-35 cm, 2 35-40 cm and >40 cm) along 3 replicate 5x50 m belttransects for fish >10cm and 3 replicate 2x50 m2 area for fish <10cm. Data was converted to number of -1 individuals per hectare (no.ha ) (fish abundance) and -1 also kilograms per hectare (kg.ha ) (fish biomass) using length-weight relationships for fish species using the following equation:

W=aL

W = weight (kg) a and b = constant (taken from Fish Base, 2000) L = length (cm)

Figure 1. Map showing of survey sites at northern Acehnese coral reef ecosystem.

B I O D I V E R S I T A S Vol. 10, No. 2, April 2009, pp. 88-93

90 200,000 180,000

Reef fish abundance (no.ha -1)

160,000 140,000 2006

120,000

2007 100,000 80,000 60,000 40,000

Pulau Weh (Kaw asan Wisata)

Pulau Weh (Panglima Laut)

Marine Protected Area

Pulau Weh

Pasi Janeng 2

Pasi Janeng 1

Paloh

Lhoh

Leun Balee 2

Leun Balee 1

Lapeng

Lamteng

Deudap

Pulau Klah

Lhong Angin 3

Lhong Angin 2

Jaboi

Lhong Angin 1

Gapang

Beurawang

Ba Kopra

Ujung Seuke

Ujung Kareung

Sumur Tiga

Rhenteuk

Benteng

Anoi Hitam

Ujung Seurawan

Rubiah Sea Garden

Lhok Weng

Canyon

Batee Meurenon

Rubiah Channel

20,000

Pulau Aceh

Open Access

Figure 2. Mean abundance (± SE) (no.ha-1) of reef fishes in 2006 and 2007 at each study site and in four management areas.

RESULTS AND DISCUSSION In 2006, reef fish abundance was highest in Sumur -1 Tiga (133,050 no.ha ), while in 2007 it was highest at -1 Benteng (83,770 no.ha ) (Figure 2). Both sites were located within Panglima Laot management areas. Although the abundance of reef fishes was significantly affected by site (nested within management), the effects of management had a greater influence of fish abundance because of more fish within marine managed areas within Panglima Laot sites than open access sites (F=2.586; P<0.05) the effects of management had a greater influence of fish abundance (i.e. Higher F ratios; F=37.671; P<0.005). Management controls that reduce pressure particularly on carnivorous and herbivorous reef fish species are urgently required to maintain reef diversity from having negative impacts on coral reefs. The significant effect of time on fish abundance was due to a decrease in overall fish numbers from -1 -1 2006 (32,788 no.ha ± 4,313) to 2007 (17,986 kg.ha ± 1,636). This decrease in fish abundance was due to lower numbers of Pomacentrids (Figure 3), which comprised much of the small size fishes (5-10 cm) that also decreased in numbers from 2006 to 2007. The key finding of the 2007 survey was that reef fish abundance in marine managed areas was considerably better than in areas open to unregulated fishing activity on Weh Island. The low cover of live corals in the Aceh Islands indicates that the coral

reefs of these islands have been heavily degraded by destructive fishing activities. These reefs also contain high numbers of macro-invertebrates, in particular sea urchin populations, that may be have been caused by the removal of predators by overfishing, and also from nutrient runoff from poorly located and managed agricultural development (Baird et al., 2005; Rudi et al., 2008). This result is supported by Wantiez et al. (1997) and Aswani et al. (2007) that species richness, density and biomass of reef fish on the protected reefs was higher and statistically significant compare with open access reef. Pomacentrids were the most abundant reef fish present on these reefs and a major prey item for large carnivorous fishes, such as groupers, snappers, and jacks. The increasing numbers of carnivorous fish between 2006 and 2007 and increasing numbers of large size fishes (20-25 cm) which consisted mostly of groupers, snappers, and jacks (unpublished data) suggests that predation may be responsible for the overall decrease in fish abundance. Reduced fishing pressure following the tsunami in 2005 may have contributed to the increased numbers of large carnivorous fish. In contrast, there was no significant different in reef fish biomass between 2006 and 2007 (F= 2.495; P>0.005). Biomass of reef fishes in 2006 ranged from -1 118 to 2399 kg.ha . The highest was recorded at Sumur Tiga, and the lowest was at Pasi Janeng2. In 2007, biomass of reef fishes ranged from 149 to 1562

RUDI et al. – Reef fish status in Northern Acehnese

80,000

Reef fish abundance (no.ha

-1)

70,000

60,000 ACANTHURIDAE

50,000

CHAETODONTIDAE LABRIDAE

40,000

POMACANTHIDAE POMACENTRIDAE

30,000

SCARIDAE

20,000

10,000

Pulau Weh (Kaw asan Wisata)

Pulau Weh (Panglima Laut)

Pulau Weh

Marine Protected Area

Pulau Aceh

Pulau Weh (Kaw asan Wisata)

Open Access

Pulau Weh (Panglima Laut)

Pulau Weh

Marine Protected Area

2006

Pulau Aceh

Open Access 2007

-1

Figure 3. Mean (+SE) of abundance (no.ha ) of reef fishes of 2006 and 2007 at each study site and type of management based on six major families that commonly found.

3,500

Reef fish biomass (kg ha -1)

3,000

2,500

2,000

2006 2007

1,500

1,000

Pulau Weh (Kaw asan Wisata)

Pulau Weh (Panglima Laut)

Marine Protected Area -1

Pulau Weh

Open Access

Figure 4. Mean (± SE) of biomass (kg.ha ) reef fish of 2006 and 2007 at each site and four management areas.

Pasi Janeng 2

Pasi Janeng 1

Lhoh

Pulau Aceh

Paloh

Leun Balee 2

Lapeng

Leun Balee 1

Lamteng

Deudap

Pulau Klah

Lhong Angin 3

Lhong Angin 2

Lhong Angin 1

Jaboi

Gapang

Beurawang

Ba Kopra

Ujung Seuke

Ujung Kareung

Rhenteuk

Sumur Tiga

Benteng

Anoi Hitam

Ujung Seurawan

Rubiah Sea Garden

Lhok Weng

Canyon

Batee Meurenon

Rubiah Channel

500

B I O D I V E R S I T A S Vol. 10, No. 2, April 2009, pp. 88-93

92 1,000 900

Reef fish biomass (kg ha -1)

800 700

ACANTHURIDAE CHAETODONTIDAE

600

LABRIDAE POMACANTHIDAE

500

POMACENTRIDAE SCARIDAE

400 300 200 100 Pulau Weh (Kaw asan Wisata)

Pulau Weh (Panglima Laut)

Marine Protected Area

Pulau Weh

Pulau Aceh

Open Access

Pulau Weh (Kaw asan Wisata)

Pulau Weh (Panglima Laut)

Marine Protected Area

2006

Pulau Weh

Pulau Aceh

Open Access 2007

-1

Figure 5. Mean biomass (Âą SE) (kg.ha ) of six major families reef fish in 2006 and 2007. -1

kg.ha , the highest was recorded at Canyon and the lowest at Lapeung (Figure 4). Sumur Tiga and Canyon were protected areas under the authority of Panglima Laot while Pasi Janeng 2 and Lapeng were open access areas in Aceh Islands. Reef fishes from the family Pomacentridae had the highest biomass of all sites in 2006 (Figure 5), but no change between 2006 and 2007 occurred as Acanthuridae comprised a large portion of the biomass in 2007, except within the areas under Panglima Laot where Pomacentridae was still dominant. Overall the findings show that reef fishes abundance and biomass are highest within the areas where management controls are in place, suggesting that these controls should form a part of any future coral reef management strategies proposed for the region. Although the overall abundance of reef fish declined from 2006 to 2007, the cause of this is unknown. As it was occurring at many sites it is possible that non-anthropogenic factors such as migration, season or predation may be responsible. The decrease in Pomacentridae abundance and increase in the numbers of large fishes requires further investigation. Population of carnivorous fish such as parrotfish (Family Scaridae) was low in all study sites. This indicated that northern Acehnese reef is lack of reef resilience both in managed and open access area. The consequent phase shift from coral to macroalgal dominated reefs would appear to support such predictions and even if coral recruitment does increase, the abundance of algae in this community may retard coral recovery. Parrotfish populations

have been suffering severe declines worldwide and excavating species are particularly vulnerable (e.g. Bellwood et al., 2003; Aswani and Hamilton, 2004; Ferreira et al., 2005; Floeter et al., 2006; Sabetian and Foale, 2006). As these fihes represent one of the few groups responsible for opening up new areas of reef surface for colonization, their decline would promote a shift in the nature of grazing. The harvest of excavating parrotfish individuals not only reduces grazing activity but may change the dynamics of the epilithic algal matrix, the dominant substratum cover of most reef ecosystems. Higher herbivory in the fishery closures reduced the abundance and persistence of herbivore-susceptible erect algae and created space and appropriate substratum for recruiting corals (McClanahan, 2008). The Marine Protected Area adhered to many of the current paradigms regarding effective coral reef management; local anthropogenic stressors were virtually non-existent and effective enforcement of the MPA had led to healthy populations of herbivorous fishes (Jennings et al., 1996; Ledlie et al., 2007).

CONCLUSIONS Overall the condition of reef fish abundance and biomass in marine managed areas was considerably better than in open access areas. These areas have been protected from blasting and cyanide fishing while Aceh Islandsâ&#x20AC;&#x2122; reefs have been subjected to unregulated fishing and destructive fishing. A reduction in destructive fishing activities was indicated

RUDI et al. â&#x20AC;&#x201C; Reef fish status in Northern Acehnese

by an increase in the number of carnivorous fish from 2006 to 2007, such as groupers, snappers, and jacks which are target species for local fisheries. Higher numbers and abundance of reef fish in marine managed areas, such as Panglima Laot in Sabang, could be considered for further conservation purpose of marine resources, especially in Nanggroe Aceh Darussalam Province. Further studies and monitoring are required to examine if recovery of Aceh Island reefs continues and compare these trends with nearby reefs where existing management controls on fisheries also require support and strengthening.

ACKNOWLEDGEMENTS Authors would like to thanks IUCN-CORDIO (Coastal Oceans Research and Development-Indian Ocean) for funding. Thanks also to Director of Marine Science Center of Syiah Kuala University; Dr. Hansa Chansang from Phuket Marine Biological Centre (PMBC) Thailand; and for U-Dive Shop at Sabang, Aceh.

REFERENCES Allen, G.R., and M. Adrim. 2003. Coral reef fish of Indonesia. Zoological Studies 42: 1-72. Ardiwijaya, R.L., T. Kartawijaya, Y. Herdiana, and F. Setiawan. 2007. The Coral Reefs of Northern Aceh: an Ecological Survey of Aceh and Weh Islands, April 2006. Bogor: Wildlife Conservation Society-Marine Program Indonesia. Aswani, S., and R.J. Hamilton. 2004. Integrating indigenous ecological knowledge and customary sea tenure with marine and social science for conservation of bumphead parrotfish (Bolbometopon muricatum) in the Roviana Lagoon, Solomon Islands. Environmental Conservation 31: 69-83. Aswani, S., S. Albert, A. Sabetian, and T. Furusawa. 2007. Customary management as precautionary and adaptive principles for protecting coral reefs in Oceania. Coral Reefs 26:1009-1021 Baird, A.H., S.J. Campbell, A.W. Anggoro, R.L. Ardiwijaya, N. Fadli, Y. Herdiana, T. Kartawijaya, D. Mahyiddin, A. Mukminin, S.T. Pardede, M.S. Pratchett, E. Rudi, and A.M. Siregar. 2005. Acehnese reefs in the wake of the Asian Tsunami. Current Biology 16:1926-1930 Bellwood, D.R., A.S. Hoey, and J.H. Choat. 2003. Limited functional redundancy in high diversity systems: resilience and

ecosystem function on coral reefs. Ecological Letter 6: 281285. Brown, B.E. 2005. The fate of coral reefs in the Andaman Sea, eastern Indian Ocean following the Sumatran earthquake and tsunami, 26 December 2004. The Geographical Journal 171: 372-374 Campbell, S.J., R.L. Ardiwijaya, S.T. Pardede, Y. Herdiana, A. Mukminin, T. Kartawijaya, A.W. Anggoro, A.M. Siregar, N. Fadli, and A.H. Baird. 2005a. Impacts of the Tsunami on Coral Reefs in the Northern Aceh Region-A report to USAID Coastal Resource Management Project II. Bogor: WCS-Marine Program, Indonesia. Campbell, S.J., M.S. Pratchett, A.W. Anggoro, R.L. Ardiwijaya, N. Fadli, Y. Herdiana, T. Kartawijaya, D. Mahyiddin, A. Mukminin, S.T. Pardede, E. Rudi, A.M. Siregar, and A.H. Baird. 2005b. Disturbance to coral reef in Aceh, Northern Sumatra: impacts of the Sumatra-Andaman tsunami and pre-tsunami degradation. Atoll Research Bulletin 544: 55-78. English, S., C. Wilkinson, and V. Baker. 1997. Survey Manual for Tropical Marine Resources. Townsville: Australian Institute of Marine Science. Ferreira, C.E.L., J.L. Gasparini, A. Carvalho-Filho, and S.R. Floeter. 2005. A recently extinct parrotfish species from Brazil. Coral Reefs 24:128-134. Floeter, S.R., B.S. Halpern, and C.E.L. Ferreira. 2006. Effects of fishing and protection on Brazilian reef fishes. Biological Conservation 128: 391-402. Hill, J., and C. Wilkinson. 2004. Methods for Ecological Monitoring of Coral Reefs. Townsville: Australian Institute of Marine Science. Jennings, S., S.S. Marshall, and N.V.C. Polunin. 1996. Seychellesâ&#x20AC;&#x2122; marine protected areas: comparative structure and status of reef fish communities. Biological Conservation 75: 201-209. Ledlie, M.H., N.A.J. Graham, J.C. Bythell, S.K. Wilson, S. Jennings, N.V.C. Polunin, and J. Hardcastle. 2007. Phase shifts and the role of herbivory in the resilience of coral reefs. Coral Reefs 26: 641-653 McClanahan, T.R. 2008. Response of the coral reef benthos and herbivory to fishery closure management and the 1998 ENSO disturbance. Oecologia 155: 169-177 Randall, J.E. 1998. Zoogeography of shore fishes of the IndoPacific region. Zoological Studies 37: 227-268. Rudi, E. 2005. Kondisi terumbu karang di perairan Sabang Nanggroe Aceh Darussalam setelah tsunami. Ilmu Kelautan 10: 50-60 Rudi, E., S.A. Elrahimi, S. Irawan, R.A. Valentino, Surikawati, Yulizar, Munandar, T. Kartawijaya, Y. Herdiana, F. Setiawan, S. Rizal, S.T. Pardede, and S.J. Campbell. 2008. Post Tsunami Status of Coral Reef and Fish in Northern Aceh, CORDIO Report. Banda Aceh: Unsyiah-IUCN /CORDIO-WCS. Sabetian, A., and S. Foale. 2006. Evolution for artisanal fisher: case studies from Solomon Islands and Papua New Guinea. SPC Traditional Marine Resources Management Knowledge Information Bulletin 20: 3-8. Wantiez, L., P. Thollot, and M. Kulbicki. 1997. Effects of marine reserves on coral reef fish communities from five islands in New Caledonia. Coral Reefs 16: 215-224.

BIODIVERSITAS Volume 10, Number 2, April 2009 Pages: 94-97

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100207

Characterizing Social Interactions and Grouping Patterns of Sumatran Orangutans (Pongo abelii) in the Gunung Leuser National Park, Sumatra JITO SUGARDJITO

♥

Zoology Division, Research Center for Biology, Indonesian Institute of Sciences (LIPI), Cibinong-Bogor 16911. Received: 3rd January 2009. Accepted: 16th March 2009.

ABSTRACT The character of social interactions and grouping patterns of Sumatran orangutans (Pongo abelii) have been studied in Ketambe research station of the Gunung Leuser National Park, Sumatra. A total number of 141 groupings and 47 incidences of interactive behavior were observed during the course of study. The character of groups (feeding group or travel-band) and the type of food trees (fig tree or fruit tree) appear to influence the interactive behavior of individual orangutans. Intolerance behavior has been characterized by feeding group in large fig trees, whereas tolerance and sexual behavior were shown mostly in travel-band. © 2009 Biodiversitas, Journal of Biological Diversity Key words: feeding group, travel-band, social interaction, Sumatran orangutan, Pongo abelii.

INTRODUCTION Orangutan is one of the largest arboreal frugivorous animals. Male of orangutans weigh up to 84 kg whereas female average about 38 kg (Rodman, 1984). In contrast to many other species of primates, orangutan lives in semi solitary life. Aggregations, however, have occurred occasionally and usually happen when they feed in large food patches or in sexual context (MacKinnon, 1974; Schurmann and van Hooff, 1986). All studies of wild orangutan have shown that more than 60 percents of orangutans’ food are fruits (Rodman, 1973; Rijksen, 1979; Galdikas, 1979). Previous study of grouping in wild orangutans has revealed that two types of groups can be distinguished i.e. feeding group (aggregations of orangutan in fig trees) in which individuals feed together within a large fig tree and leave separately whereas travel-band in which individuals feed together within a patch of fruit trees, leave together, stay together for some time, and visit the next fruit patch together (Sugardjito et al., 1987). Despite the fact many field studies over the past 30 years have produced a wealth of data about orangutans, little is known about sociality of wild orangutans. Intensive studies of the sociality and

 Correcponding address: Jl. Raya Jakarta-Bogor Km 46, Cibinong 16911 Tel. & Fax.: +62-21-8765056 & 8765068 e-mail: jitos@cbn.net.id

sexual behavior of this animal have been conducted mostly in captivity (Edward, 1982; Nadler, 1982). There were only few data available on the sociality of wild orangutans (Galdikas, 1985; Utami, 2000). However, data on the associations between sociality and group formations in wild orangutan are lacking. By characterizing social interactions and grouping patterns, therefore, we may be better able to understand the relationships between interactive behavior and its ecological determinants which are important for the future conservation needs of orangutan, particularly when we reintroduce a group of orangutans in the wild. This paper presents the association between grouping patterns and social interactions which is shown when orangutans formed a group either in fig trees or in fruit bearing trees.

MATERIALS AND METHODS Study area The field work was carried out in the Ketambe research station of the Gunung Leuser National Park, Sumatra (Figure 1). The study area is covered by mixed lowland tropical rain forest which is characterized by a high diversity of fig tree species, while the fruit trees species mostly belong to the family Meliaceae and Euphorbiaceae (Abdulhadi et al., 1984). The majority of large fig trees are strangler with very wide crown and fruiting asynchronously, on individual schedule (Schaik, 1996)

SUGARDJITO â&#x20AC;&#x201C; Social interactions and grouping of Pongo abelii

Procedures The population of Sumatran orangutans (Pongo abelii) in the area has been studied continuously since 1971 (Rijksen, 1978; Schurmann, 1981; Sugardjito, 1986; Utami, 2000). Seven habituated animals (1 adult male, 3 adult females, 2 adolescent females, and 1 adolescent male) were followed for 3 up to 10 days from dawn to dusk from February 1980 until December 1982. Data on interactive behavior was collected when focal animals were part of a group and sampled in a one-zero score (Altman, 1974). The group definitions were following Sugardjito et al. (1987). We sampled only types of interactive behavior that could be clearly observed, e.g. displacing, playing, sharing of food (tolerance to other individuals when the food is taken), and sexual interactions (copulation and genital investigation). These types of interactive behaviors have been classified into three categories, namely intolerance, tolerance, and sexual. The data were analyzed statistically with a Chi-square and Fisher tests for two or three dimensional contingency tables (Siegel, 1956; Everitt, 1977).

RESULTS AND DISCUSSION A total number of 141 groupings were observed during the course of study. Feeding groups occurred mainly in large fig trees while travelbands can be formed in fruit trees other than fig trees (Table 1). Forty-seven incidences of interactive behavior were observed, and all of these have been characterized into three behavioral categories (Table 2). Regardless whether orangutan forms the group in fig trees or fruit trees, no differences were found in the frequencies of appearance of these three behavioral categories. It means that orangutan potentially shows the interactive behavior in equal opportunity while feeding in either fig or fruit trees. However, when these behavioral categories are partitioned according to the food tree types (figs or fruits) then the differences were observed (Table 3). The intolerance behavior appeared almost exclusively in large fig trees. In contrast, the tolerance and sexual interactions were observed particularly in fruit trees in which the travelbands were formed. The data also showed that intolerance behavior was associated with the combined category e.g. feeding group in fig trees. As for the other two behavior categories did not (Table 3). These two behavior categories could be found mostly in travel-band.

Figure 1. Location of the study area in the Gunung Leuser National Park, Sumatra.

B I O D I V E R S I T A S Vol. 10, No. 2, April 2009, pp. 94-97

Table 1. The grouping incidences of orangutan while feeding in fig trees and fruit trees in Ketambe area of 2 Sumatra. (X = 19.78, df=1, p<0.01) Group formations Feeding group Travel-band

Fig tree

Fruit tree

Total

66 25

17 33

83 58

Table 2. The frequency of interactive behavior incidences of 2 orangutans based on three categories. (X =5.39, df=2, n.s.).

Number of incidences

Behavioral interactions Intolerance Tolerance Sexual Total 16 22 9 47

Table 3. The frequencies of each condensed interactive behavioral category of Sumatran orangutans with respect to 2 the social conditions and types of food trees. (X =39.13, df = 7, p<0.001) Social Travel-band Feeding group Total interactions Fig tree Fruit tree Fig tree Fruit tree Intolerance 0 2 13 1 16 Tolerance 6 10 4 2 22 Sexual 3 5 1 0 9

The heavy arboreal orangutans require a large amount of calories daily (Wheatley, 1982). This factor combined with calorie content of figs and the enormous size of fig trees led to argue that figs are an important staple food for orangutans. Sugardjito et al. (1987) showed that orangutans visit large fig trees in much more constant fashion throughout the year than the other fruit trees. Since large fig trees are relatively less in number compared to the other fruit trees in the area (Schaik, 1996), it will force the orangutan to return regularly to the same individual fig tree. Therefore the favorite and relatively rare fig trees could attract aggregations of orangutans for nutritional reasons. The spreading crown of a single large fig tree could reach about 30 meters, whereas the wide crown of small-medium fruit trees only about 5 meters. Further, these small-medium fruit trees usually grow abundantly in a large patch of forest whereas the large fig trees occur only a few at a rather distances. Orangutans, therefore, visit many species of fruit trees roughly as expected on the basis of their abundance in the forest. This suggests that orangutansâ&#x20AC;&#x2122; strategy in harvesting fruiting trees is opportunistic, i.e. in proportion to availability. However, the frequency in which fig trees are visited is far higher than expected on this basis. It is rare to have several individual of the same species of large fig trees in fruit at the same time. This is in contrast to the small-medium fruit trees. These trees are synchronously, it is often to have several individuals of the same species bearing fruits together at the

same time. Further, the median feeding time per visit in a fig tree is longer than in a fruit tree (Sugardjito, 1996). This could be affected by the number of orangutan aggregates in a fig tree. Indeed, Utami (2000) has found that orangutan spent more time feeding when aggregation size increased in a fig tree. Consequently, the intolerance interactions would appear more frequent between the dominant individual and subordinate individuals in a fig tree. The differences of growing pattern, the density and fruit bearing availability between fruit trees and fig trees would have different effects on the social interactions of orangutans. When a group of orangutan was feeding in a large fig tree, it has been shown that the availability of ripe fruit was positively related to the number of orangutans visiting a fig tree and their foraging efficiency (Utami, 2000). This might lead to intolerance behavior between individuals to be associated with feeding group in large fig trees. This phenomenon did not exist when orangutan forms a travel-band in small-medium size fruit trees. Due to the relatively smaller size of fruit tree compared to fig tree, the fruit tree provides only limited space and therefore, a single fruiting tree could hold only 2 to 4 orangutans. The other member of group of individual orangutans will exploit the neighboring fruit trees which occur in a large patch and clumped. This suggests that individuals feeding together in a fruit tree are belonging to the same social group and hence it reduces intolerance behavior between individuals.

CONCLUSIONS The findings imply that both forms of aggregations and the growing patterns of food trees (figs and fruits) would determine the social interactions of wild orangutan. The intolerance behavior is associated with feeding groups in large fig trees, whereas the tolerance behavior and sexual interaction did not. The growing patterns of small-medium size fruit trees which grow abundantly in large patch and clusters could reduce intolerance interactions between orangutans while feeding. This is in contrast to a few huge fig trees which grow scarcely and the availability of figs are on individual schedule would attract many orangutans visit in the same fig tree. Consequently, it could lead the dominant orangutan performs intolerance behavior to its subordinate.

ACKNOWLEDGMENTS I thank Prof. Dr. J.A.R.A.M. van Hooff and Dr. I.J.A. te Bookhorst of the University of Utrecht for their constructive discussions when developing this paper. I am also grateful to the late Nina Sulaiman of the Universitas Nasional, Jakarta who made possible for the author to do field work. I thank the Directorate of

SUGARDJITO â&#x20AC;&#x201C; Social interactions and grouping of Pongo abelii

Forest Protection and Nature Conservation for allowing the author to work in the Gunung Leuser National Park. The author benefited from facilities provided by Fauna and Flora International-Indonesia Programme for revisiting and analyzing the data. This study has been funded by the Dutch Ministry of Education and Science through the University of Utrecht and Universitas Nasional, Jakarta. Finally, I appreciate Prof. Dr. Ali Saad Mohamed for reviewing and editing the manuscript.

REFERENCES Abdulhadi, R., K. Kartawinata and R. Yusuf. 1984. Structure and composition of forest pattern in the Ketambe area, G. Leuser National Park. Project Report to LBN-LIPI. Bogor: Lembaga Biologi Nasional, LIPI (in Bahasa Indonesia). Altman, S.A. 1974. Baboons, space, time and energy. American Zoology 14: 221-248. Edward, D.S. 1982. Social potential expressed in captive group living orangutans. In: de Boer (ed.). The Orangutan, its Biology and Conservation. The Hague: Junk. Everitt, B.S. 1977. The Analysis of Contingency Tables. London: Chapman and Hall. Galdikas, B.M.F. 1979. Orangutan adaptation at Tanjung Putting Reserve, mating and ecology. In: Hamburg, D.A. and E.R. McCown (eds.). The Great Apes. Menlo Park, CA.: Benjamin/Cummings. Galdikas, B.M.F. 1985. Orangutan sociality at Tanjung Putting. American Journal of Primatology 9: 101-119. MacKinnon, J. 1974. The behavior and ecology of orangutans (Pongo pygmaeus). Animal Behaviour 22: 3-74. Nadler, R.D. 1982. Reproductive behavior and endocrinology of orangutans. In: de Boer (ed.). The Orangutan, its Biology and Conservation. The Hague: Junk. Rijksen, H.D. 1978. A Field Study on Sumatran orangutans (Pongo pygmaeus abelii Lesson 1827). Wageningen: H. Veenman and Zonen B.V.

Rodman, P.S. 1973. Population composition and adaptive organization among orangutrans of the Kutai Nature Reserve. In: Michael, R.P. and J.H. Crook (eds.). Ecology and Behavior of Primates. London: Academic Press. Rodman, P.S. 1984. Foraging and social systems of orangutans. In: Hamburg, D.A. and E.R. McCown (eds.). The Great Apes. Menlo Park, CA.: Benjamin/Cummings. Schaik, C.P.van. 1996. Strangling figs: Their role in the forest. In: Schaik, C.P. van and J. Supriatna (eds.). Leuser: A Sumatran Sanctuary. Jakarta: Yayasan Bina Sains Hayati. Schurmann, C.L. 1981. Courtship and mating behavior of wild orangutans in Sumatra. In: Chiarelli, A.B. and R.S. Corruccini (eds.). Primate Behaviour and Sociobiology. Berlin: SpringerVerlag, Schurmann, C.L. and J.A.R.A.M. van. Hooff, 1986. Reproductive strategies of the orangutan: new data and reconsideration of existing socio-sexual models. International Journal of Primatology 7: 265-287. Siegel, S. 1956. Non-parametric Statistics for the Behavioral Sciences. Kogakusha, Tokyo: McGraw Hill. Sugardjito, J. 1986. Ecological Constraints on the Behavior of Sumatran Orangutan (Pongo pygmaeus abelii) in the Gunung leuser National Park, Sumatra. [Ph.D. Dissertation]. Utrecht: University of Utrecht. Sugardjito, J. 1996. The behavioral ecology of Sumatran orangutans. In: C.P.van Schaik and J. Supriatna, (eds.). Leuser: A Sumatra Sanctuary. Jakarta: Yayasan Bina Sains Hayati. Sugardjito, J., I.J.A. te Boekhorst, and J.A.R.A.M. van Hooff. 1987. Ecological constraints on the grouping of wild orangutans (Pongo pygmaeus) in the Gunung Leuser National Park, Sumatra, Indonesia. International Journal of Primatology 8: 1741 Utami, S.S. 2000. Bimaturism in Orang-utan Males: Reproductive and Ecological Strategies. [Ph.D. Dissertation]. Utrecht: University of Utrecht. Wheatley, B.P. 1982. Energetic of foraging in Macaca fascicularis and Pongo pygmaeus and a selective advantage of large body size in the orangutan. Primates 23: 343-368.

BIODIVERSITAS Volume 10, Number 2, April 2009 Pages: 98-103

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100201

Cultivation Practices and Knowledge of Local Rice Varieties among Benuaq Farmers in Muara Lawa District West Kutai, East Kalimantan-Indonesia 1,2,♥

MEDI HENDRA

2,3

, EDI GUHARDJA , DEDE SETIADI , EKO BAROTO WALUJO , YOHANES PURWANTO

Biology Department, Faculty of Mathematics and Natural Sciences, Mulawarman University (UNMUL), Samarinda 75123 2 Post Graduate School, Bogor Agricultural University (IPB), Bogor 16680 3 Biology Department, Faculty of Mathematics and Natural Sciences, Bogor Agricultural University (IPB), Bogor 16680 4 Laboratory of Ethnobotany, Research Center for Biology, Indonesian Institute of Sciences (LIPI), Cibinong-Bogor 16911 Received: 4th February 2009. Accepted: 21st March 2009.

ABSTRACT This research aim to study how Benuaq society around Muara Lawa District, manage agriculture system and internal knowledge about rice varieties. This research use ethnobotanical approach: collecting ethnobotanical data of rice varieties and traditional system of swidden cultivation. Data was obtained by using direct participatory technique by interviewing the people (key informants) about their practice and perception. The interviews were unstructured open-ended discussion on knowledge and farming activities including about diversity in rice varieties. Subsequently, quantitative data from questionnaires was combined with depth-interview data from key informants. The Benuaq indigenous systems, practices, and cultivation preferences held by the Benuaq are guarded toward maintaining their rice diversity. The Benuaq systems of upland rice cultivation, site selection for umaq establishment, plant species for fertility indicator, and cultivation of wide ranges of upland rice varieties indicate their sophisticate knowledge in agriculture. Unfortunately there has been degradation in the indigenous knowledge among the young Benuaqs. © 2009 Biodiversitas, Journal of Biological Diversity Key words: Benuaq, ethnobotanical, indigenous, upland rice.

INTRODUCTION Benuaq is one of the Dayaks in Borneo, particularly the Indonesian part (i.e. Kalimantan). They settled in the southern tributaries of the great Mahakam River, namely the Bongan, Ohong, Jelau, Kelawit, Tuang, Lawa, Pahu and Nyuwatan (all in the Indonesian province of East Kalimantan) as well as in the upper Teweh (Central Kalimantan Province). They are profoundly concentrated in some regions in the West Kutai District, such as Barong Tongkok, Bentian, Bongan, Damai, Muara Lawa, Muara Pahu and Siluq Ngurai. Despite the information that the Benuaq tribe has long been regarded as belonging to the larger Lawangan group (Riwut, 1979). Massing (1981) mentioned that the Benuaq are rarely cited in the literature on the Dayaks of Borneo, thus it has been regarded as the least known tribe.

 Corresponding address: Jl. Barong Tongkok, Kampus UNMUL G.unung Kelua, Samarinda 75123 Tel: +62-541-749140, 749152. Fax: +62-541-749140 e-mail: medihendra @yahoo.com

Nevertheless, the Benuaq are the largest Dayak group in the West Kutai district in the Indonesian province of East Kalimantan and have preserved their tradition and customs (“adat”) more than any other tribes. Seeland and Schmithusen (2002) reported that Benuaq Dayak has combined the shifting cultivation and cultivated forest products as their livelihood base. This proves their deep understanding on their surrounding natural resources. The importance of Borneo in the diversity of rice varieties has been recognized by both anthropologists and agriculturists (Sutlive, 1978; Padoch, 1988; Setyawati, 1997). Freeman (1970) and Padoch (1988) reported that Dayak farmers are able to identify and cultivate various varieties of rice. This finding is in accordance with the results of Watson (1984), Fujisaka (1987) and Damus (1995) in some recent studies that traditional farmers in many places in the world are capable in maintaining the genetic diversity of rice varieties in order to match the local environmental conditions. Unfortunately, such studies were rarely gone below the level of the local communities. Despite gathering information concerning the total number of rice varieties known to the community, such researches (such as by Widjaja and Jessup,

HENDRA et al. – Local rice varieties of Benuaq farmers

1986) failed to provide an information on the number of varieties that each individual farmer actually planted. This failure raised awareness among agriculturists and anthropologists, who warned that changing economic and environmental conditions may cause loss in germplasm biodiversity, particularly in the genetic resource areas. Swidden agriculture has been practiced in East Kalimantan for a very long time and it was suspected as a cause of establishment of alang-alang grassland (Imperata cylindrical Beauv.) ± 400.000 ha at present and secondary forest presently 2.4 million ha (Kartawinata et al. 1981). In rotational systems, the length of cultivation and fallow periods, and the swiddening techniques determine the development and structure of swidden fallow secondary forest (Schmidt-Vogt, 2001). According de Jong et al. (2001) swidden fallow secondary forest defines as “forest regenerating largely through natural processes in woody fallows of swidden agriculture for the purposes of restoring the land for cultivation again”. The current research was carried out in the Benuaq community of Muara Lawa District, East Kalimantan, Indonesia for six months. Data were collected through five weeks of fieldworks from June to December 2008 proceeded in three villages Dingin, Muara Lawa and Lambing. Data collecting was focused on four specific questions: (i) Why do the farmers select and maintain certain rice varieties? (ii) Do some of the farmers maintain certain varieties that were abandoned by other farmers? (iii) Is the traditional practices effective in conserving the biodiversity? (iv) How are the knowledge of varieties and the seeds themselves disseminated? This current study also attempted to understand the operating mechanisms in the acquisition and maintenance of rice varieties among farmers following the previous example by Vayda (1992).

MATERIALS AND METHODS This current research was focused on individual knowledge and behavior in farming activities and obtaining explanations for the occurrence of genetic diversity in rice varieties and the dynamics of farmer’s knowledge of rice. The ethnobotanical approach was implemented involving collecting data on rice varieties and traditional system of shifting cultivation. Data was obtained using direct participatory technique through interview with the important participants (key informants) regarding their practices and perceptions. The interviews were unstructured open-ended discussion on their knowledge and farming activities including information on the diversity of rice varieties. Two hundreds households of approximately 1000 farmers of shifting cultivation in three areas were sampled. The households selected were those, in which farming is the main livelihood and shifting agriculture have been made in the previous three years.

Quantitative data from questionnaires were combined with depth-interview data from key informants. The interviews cover two important aspects: (i) Shifting cultivation, which includes numbers of rice fields during the last three years and ownership, plants indicator of fertility, land topography and soil characteristics, succession stages, rice cultivation steps, rice growth level, and quantity of harvest. (ii) Knowledge of rice varieties, which covers rice varieties, sources of seeds and individual knowledge. Information obtained then was combined with literature study, ethnobotany of rice varieties, and the local knowledge of environment.

RESULTS AND DISCUSSION Land-space selection basis and preparatory stages The result of this current study indicates that the Benuaq cultivates upland rice on annual field (umaq). This annual cycle of rice cultivation is the centre of life in the Benuaq societies in the West Kutai district. Among respondents 74.44% own 1 to 2 hectares of annual swidden area, 15.93% less than 1 hectare and only 9.63% more than 2 ha (Table 1). Table 1. Areas of annual swidden (umaq) own by the Benuaq people in Muara Lawa District on planting season of 2006/2007. Areas of annual swidden Less than 1 hectares 1 -2 hectares More than 2 hectares Total

Respondents (N)

43 201 26 270

15.93 74.44 9.63 100

The selection of a suitable site to establish field is carefully and involving consultations and cautious observations. After performed, the shifting cultivation activities are started. These activities are usually started by land clearing in June or July to minimize pests’ threats. The future rice fields of different households are planed close to each other. Recently the clearing of primary forest has become very rare as the forest located more distant from village. Tanyut (honey trees) as Aput (Dipterocarpus spp.), Itir (Intsia palembanica Miq.), Jelemuq (Canarium decumanum Gaertn.), Jengan (Shorea laevis Ridley), Kawang (Shorea seminis (De Vriese) Sloot.), Lelutukng metapm (Alstonia pneumatophora Backer), Merjaakng (Sindora leiocarpa Backer), Ngoiq (Dryobalanops lanceolata Burck.), Puti (Koompassia excelsa (Becc.) Taub.), Tempudou (Dipterocarpus confertus Sloot.), dan Tudak (Artocarpus sp) and potential trees (e.g. fruit trees) within the rice field areas are not cleared. Bordering fields are cleared (ladekng) and ignitable materials are avoided in order to prevent unintended fires invading the neighboring fields or forests. Soon after the burning field was hand planted. This is normally done around September.

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When rice has grown up reaching knee height vegetables, cassava, banana, chili, sweet potato, eggplant and other perennial crops are planted. The plots are weeded regularly and rice is harvested around March in the following year, while other crops are left to mature. The result of this current research shows that the Benuaq is familiar with the fallow land fertility indicator. The fertility level is checked using certain plants as indicators (Table 2). The varieties of rice planted vary considerably according to the land need and type. Based on land topography and soil characteristic the Benuaq farmers distinguish land farming into 3 types: (i) Dempaak, which are flatlands between two hills. (ii) Kerebeek or kerereng, which are sloping land but less steep. It is the most commonly used land for rice. (iii) Payaakq, which are wet or swampy areas used for wet-rice varieties. Payaakq is divided into two subtypes: (a) Common payaakq, which is smooth land at lowland level and always soaked with water. (b) Payaakq belikuq engkok, which is unsmooth land at lowland level. Only half of the area is soaked with water, the rest is dry land. According to soil characteristics (color and structures) the Benuaqs distinguish four types of soil: (i) Tana metapm, which means black soil. (ii) Tana meaq, which means red soil. (iii) Tana ronan, which means clay soil. (iv) Tana one, which means sandy soil. Tana one then is divided into three subtypes: (a) Tana one lemit (yellow sandy soil), which is found in riverbank. (b) Tana one metapm (black sandy soil), which is found far from riverbank. This type of soil is fertile with degree of fertility based on land succession stage. (c) Tana one bura (white sandy soil) usually infertile land kerangas. Other classification of lands is based on features such as the plant species present. The Benuaqs understood that slash-and-burn events influence the time scale of the subsequent development of each area of fallow land. They use this knowledge to plan their next slash-and-burn event. The result of this current study indicates that

base on the differences in the succession stages the Benuaq classify the fallow lands into: (i) The initial stage of secondary growth (aged 1-3 years) is called urat and consisting shrubs and herbs. (ii) The young secondary forest which follows urat and is called balikng bataakng (aged 5-15 years) and consisting of pioneer tree species. (iii) The old secondary forest (aged 20 to 30 years) or is called bataakng. (iv) The older stage forest which is called bengkal tuhaq (particularly when over-45 years old). This result is in accordance to Okimori and Matius (2000). The Benuaq farmers have also discovered and maintained a swiddens working system with a certain periodicity. The result of this current study regarded as the most appropriate farming method for local conditions and the economically most profitable. The cycles of shifting cultivation the Benuaqs was presented in the Figure 1. Rice is a main crop in Benuaq farmers field (umaq). It is both praised as both staple food and a cultural representation that determines their way of life. This is accordance with Sellato (1989) that rice is source of entire life aspects for Benuaqs. The Benuaqs rice harvesting cycle is honored with taboo and ceremonies. The Benuaqs categorize their swidden area based on the growth level of rice into 14 levels (Table 3). The time to maturity for upland rice is about 4 to 6 months depending on the varieties. The Benuaqs use ex-container (i.e. can) of cooking oil to count the crops, in which one equals to 10 kg of dry paddy. One hectare dry field produces 100 to 250 cans or approximately 1 to 2,5 tons of dry paddy. Part of the harvested crops is stored in kelengkikng (rice barn), while other part is sold for cash. Harvest usually decreases in the following year, thus farmers move into another land in order to get a fertile clearing land. The Benuaqs acknowledge that their rice productivity is somehow closely connected with the land fertility and in turn the land fertility is related with the stage of forest succession; thus the soil in the previous fields have to be given enough time to recover its fertility.

Table 2. The fertility indicator plants at Benuaq society in Muara Lawa. Local name Bateteq Belaban Belayatn Bengkuukng Beramboyut Biruq Butootn Isaaq Jaung Kayu sirih Nagak Pipit Pisaaq Tempuro Tentakng

Scientific name Hornstedtia sp. Tristaniopsis whiteana (Griff.) Wilson & Waterhouse Merremia sp. Macaranga gigantea (Reichb. f. & Zoll.) Mull. Arg. Macropanax dispermus Bl. Licuala valida Becc. Stachypterinum sp. Cominsia gigantea K. Schum. Nicolaia speciosa (Bl.) Horan Piper aduncum L. Schima wallichii (D.C.) Korth. Lithocarpus elegans (Bl.) Hatus. Ex S. Fordia splendidissima Bl. Dillenia sp. Campnosperma auriculatum (Bl.) Hook. f.

Family Zingiberaceae Myrtaceae Convolvulaceae Euphorbiaceae Cucurbitaceae Arecaceae Maranthaceae Maranthaceae Zingiberaceae Piperaceae Theaceae Fagaceae Fabaceae Dilleniaceae Myrsinaceae

HENDRA et al. – Local rice varieties of Benuaq farmers

Heaps are burnt Rituals (kerongo)

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Use a dibble Collectively

Concentrically Overall surface is burnt

5 4

6 Periodicity of weeding

Utilization of tools

Gender (♂)

Gender (♀)

Technique

CULTIVATED AREA (UMAQ LATI TANA)

Rituals

Ceremony (Nema pare)

8 Rituals

Tool (gentuk)

2 No use fences

Deposited of rice

Slashed

Management of the field edges

Natural succession Agroforestry (simpukng)

Land use rights Omen Ceremonial rituals (Pakatn nyahuq)

Plantation (rubber)

Figure 1. Cycles of cultivation the Benuaqs: 1. Selecting (Nusaq lati tana), which is done a year before swidden activity. The Benuaq farmers proceed a ritual ceremony in order to identify a suitable swidden site and the most common ceremonial rituals practiced in this step is Pakatn Nyahuq (sacrifice for forest spirit). Accordance to Sellato (1989), who mentioned that most of the Dayaks select time activity based on star and the result of this current study specific star for agriculture is Bemari star, for land selecting and Piyuluq star (Pengkuluq) for sign to begin swidden activities in Benuaqs knowledge. 2. Clearing of scrubs (Nokap nerap). The area is slashed around June to July by both men and women Benuaqs. The slashed vegetations are allowed to dry under the sun for a few days for drying. 3. Felling of trees (Nowang) and cut into pieces (Nutu). It takes place around July to August. Equipment used for cut down trees are adze, axe and chopping knife. Nowadays the use of chainsaw plays an important role for time efficiency. The plot is then left to dry for a period of 4 to 5 weeks. Plots with larger trees and bigger branches require longer time for drying. 4. Burning (Nyuru) takes place at September to October. The burning is done concentrically. Ceremonial rituals is performed including creating a human picture (Kerongoq), placing it above the nyiruh (or winnowing basket for rice) and hanging it in house yard. 5. Re-burning (Mongkekng), any leftover branches and logs are collected in piles and a second burning is arranged in which both men and women get involved. 6. Sowing (Ngasaq Niruk). This kind of rice planting is done collectively. Men and women in turn are mutually and cooperatively involved pelo and beroh. Pelo is a kind of mutual cooperative in the pelo members, while beroh is workers thats helps in fields. This step is done by sowing paddy in holes into holes using a dibble (a pointed stick). 7. Weeding (Ngejikut) is grasses or weeds clearings. This work is done after rice reaches one to three months old. Before three months old, the work is intensively done in turn by women through a mutual cooperative (pelo or beroh). 8. Harvesting (Ngotepm). The ngotepm is started by a harvesting ceremony called as Nema Pare, attended only by closely related members of the house (usually one big family). This ceremony is to invite the Goddess of Paddy (Luwikng or Dewi Padi in Malay). The harvesting process is usually worked in mutual cooperative by women only that is taking turn between mothers and daughters (pelo). The small palm-held reaping knife for cutting rice stalks (gentuk) is used. The harvested paddy then is put into bamboo or rattan pouch which is tied up in waist (gamak) and carried and deposited in the rice barn (kelengkikng). 9. Fallow (Uraat-bataakng). The uraat-bataakng is the fallow stage of field (umaq) following upland rice cultivation. Uraat is advanced fallow may have been planted with various fruits, rubber and rattan plants. Bataakng is the old secondary forest of successional stage of fallow.

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Table 3. The upland rice growth level in swidden of Benuaq people. Growth level in Benuaq language Nokoq Meriwih luang asak Makur lokatn Beramaaq Untuq maih Buluq titukng Entur urakng Meetn/Belampaar Sengayo seloit Ngejatas Ngertak Lemit morakng/Luai uruk Luai tengah Luai melus

Characters The rice germinates after sowing. The seedling will grow and develop the branched tillers. The tillers growth reached 20 cm and primary tillers develop into secondary and tertiary tillers. The tillers growth and reached 40 cm high. The clump of paddy growth reached 80 cm to waist height of an adult. The clump is pointed up with mature internodes, which are hollow and finely grooved with a smooth outer surface. The panicle inflorescence is appearing from the top of the internode of the clump. The panicle inflorescence bears rice spikelets that develop into rice grains. Although the inflorescence are appearing but not yet spread evenly in the field. The spikelets are appearing in full. Half of the panicle inflorescence begins to bend as the contents (rice grains) have fully developed. Rice grains are white as milk. Filled out rice grains as legumes. Rice grains at the terminal part of the panicle infructescence are yellowing. Rice grains at the upper half part of the panicle infructescence are yellowing. The entire rice grains are yellowing this means the rice is mature, thus time for harvesting.

Farmers knowledge of rice varieties In general the Benuaqs classify their rice varieties into two: (i) Pare (the common rice), which includes 67 varieties. (ii) Pulut (the glutinous rice), which includes 36 varieties. Despite these 103 rice varieties, only few observed in this current study. Most of the varieties (97 in numbers) are for dry field farming and can be planted in various topographic conditions, while six can only be planted in wet land or swamp areas. The varieties are mostly indigenous, while few varieties were introduced from other areas. The introduced rice varieties are common in Dayak societies as was discovered by Freeman (1970) in the Ibans. The Benuaqs in Muara Lawa have detail specified terminology for categorizing their rice varieties. Each variety has a local name and the farmers are familiar with the distinctive characters or origin from which the name is derived. Various name and sub name mostly based on the original area of the name. Varieties from other areas can be combined with the local name such as pare bentiant, pare bogor or pare kenyah. Some rice varieties have connections with the myth in the Benuaq society such as pare bawiiq (bawiiq means pig in Benuaq language). The Benuaqs believe that the first grain of this rice is found in the intestine of a pig. Other example is pare tekayo (tekayo means deer in Benuaq language). Similar as in pare bawiiq, but the first grain is believed found firstly from the intestine of a deer instead. The origin of several varieties names remains unclear though. The Benuaqs prefer local varieties than introduced ones. Although the local varieties are not as productive as the introduced ones, but the indigenous varieties are more resistance to pest, more adaptive to local nutritionally poor condition, and taste better. Indeed, some farmers admitted that they do not like to cultivate the so called wetland rice simply because their family prefers the taste of upland rice.

As nowadays agriculture is in pursuit for sustainable germplasm for increasing harvest and diseases resistance, the Benuaqsâ&#x20AC;&#x2122; indigenous rice varieties open more plausible possibilities. Studies have given us important information about farmersâ&#x20AC;&#x2122; knowledge of rice varieties and some researchers have found that farmers maintain genetic diversity of rice varieties in their fields to match local environmental conditions (Fujisaka, 1987; Setyawati, 1997). Several farmers can identify or recognize the entire 103 rice varieties recorded in this research, while the others can only mention 10 to 40 varieties. In general, all farmers can recognize about 40 % of the existing local varieties. The result of this current study shows that the senior members of the Benuaq society (such as the traditional law leader or the Mantiq, his wife, and three senior farmers) have more knowledge in particular domains of expertise than younger members (Table 4). Only the Mantiq, his wife, and three senior farmers are able to identify all existing local rice varieties. Other farmers with age more than 40 years old can mention about 60 % varieties, while younger farmer with age less than 30 years old can only identify about 20 % varieties. This clearly shows that the indigenous knowledge in rice varieties in the Benuaqs is degrading. The knowledge of rice variety can also be achieved from the pelo group. In the early cultivation season, the members of pelo assemble and discuss Table 4. Average number of rice varieties known according to farmers age. Age (years) 20 -30 31 -40 41 -50 > 50 Total

Average number of varieties known 18 25 43 62

Number of respondents (N) 10 16 14 10 50

HENDRA et al. – Local rice varieties of Benuaq farmers

about the next planting season. The most important agenda is on which rice variety is to be planted for the next season. Thus, farmers develop their knowledge through experience, exchange of knowledge, and careful observation of other farmers’ plants. In each of the planting seasons, most farmers cultivate at least one variety that is different from the previous season and it depends on the harvest of the previous season. If the harvest was successful, the varieties will be cultivated again. If it was the opposite, the new varieties are implemented. Most farmer mentioned that they make plan well in advance including choosing the varieties to be planted for the next season. If the plan is to plant the same variety again, an appropriate quantity of seeds from that variety has to be separated from their harvest. If they want to try another variety for the first time, they must ask or exchange seeds with other farmers. For the Benuaqs harvesting for paddy seeds and rice grains are two different things and have to be done separately. Paddy seeds were harvested from fertile and healthy stalk, thus the quality of the rice can be maintained and increased. The instability of environmental conditions from year to year may encourage the Benuaqs farmers to try many different varieties with hope that some varieties will grow well on the changing soil condition and produce good harvest. This current study observes that occasionally the Benuaq farmers plant several different kinds of the upland rice. For example in one year planting season the families in Muara Lawa cultivate about 20 different rice varieties. This kind of rice planting is because specific variety grows better in certain environments. Success yield of each variety will be compared between harvests. If one or two varieties grow fertile after closely planted, their seeds will be harvested separately in the next planting seasons. On the contrary, if they are fall to grow, those varieties are not planted in next season. This finding is in accordance with Dove (1988) that the purpose of mix planted was to test the relative benefit of one or more rice varieties.

CONCLUSION The Benuaq Dayak indigenous systems, practices and preferences are achieved towards maintaining biodiversity. The area of Benuaq’s swidden ranged from 1 to 2 hectares, and adapted to the limitation in the manpower. The Benuaq systems of dry field rice cultivation, site selection for umaq establishment, plant species for fertility indicator, and cultivation of a wide range of upland rice varieties indicate that they have a sophisticated agricultural knowledge. There are 15 plant species for fertility indicators found. These species are used as guideline for field selection. Other guidelines include land topography and soil characteristic. The Benuaqs understood very

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well that the slash-and-burn events influence the time scale of the subsequent development of fallow lands and use this knowledge to plan their next slash-andburn event. The Benuaqs know nine steps for rice cultivation and 14 swidden rice growth levels. Farmers classify rice varieties into two: pare (the common rice) with 67 varieties and pulut (the glutinous rice) with 36 varieties and totality 103 rice varieties were recorded in Muara Lawa. Unfortunately there has been a degrading in the indigenous knowledge of rice varieties among young Benuaqs.

REFERENCES Damus, D. 1995. Pengetahuan tentang Varietas Padi dan Tipe Budidayanya pada Masyarakat Dayak Hulu Sungai Bahau. Report Culture & Conservation, Kayan Mentarang Conservation Project. Jakarta: WWF and Direktorat Jenderal Pelestarian Alam dan Perlindungan Hutan.. De Jong, W., M. van Noordwijk, M. Sirait, N. Liswanti, and Suyanto. 2001. Farming secondary forest in Indonesia. Journal of Tropical Forest Science 13 (4): 705-726. Dove, M.R. 1988. Sistem Perladangan di Indonesia: Studi Kasus dari Kalimantan Barat. Yogyakarta: Gadjah Mada University Press. Freeman, D. 1970. Report on the Iban. London School of Economics Monographs on Social Anthropology. London: The Athlone Press. Fujisaka, S. 1987. Filipino Upland Farmers: Informal Ethnoscience for Agricultural Development Research. Philipphine Studies 35: 403-409. Kartawinata, K., S. Adisoemarto, S. Riswan, and A.P. Vayda. 1981. The impact of man on a tropical forest in Indonesia. AMBIO 10: 115-119. Massing, A.W. 1981. The journey to paradise: funerary rites of Benuaq Dayak of East Kalimantan. Borneo Research Bulletin 13 (2): 85-104. Okimori, Y., and P. Matius. 2000. Tropical secondary forest and its succession following traditional slash-and-burn agriculture in Mencimai, East Kalimantan. In Guhardja, E., M. Fatawi, M. Sutisna, T. Mori, and S. Ohta (eds.). Rainforest Ecosystems of East Kalimantan: El Nino, Drought and Human Impacts. Tokyo: Springer-Verlag. Padoch, C. 1988. Agriculture in interior Borneo: shifting cultivation and alternatives. Expedition 1: 18-28. Riwut, T. 1979. Kalimantan Membangun. Jakarta: PT. Jayakarta Agung Offset. Schmidt-Vogt, D. 2001. Secondary forest in swidden agriculture in the highlands of Thailand. Journal of Tropical Forest Science 13 (4): 748-767. Seeland, K., and F. Schmithusen (eds.). 2002. A Forest Tribe of Borneo, Resource use among the Benuaq Dayak. Man and Forest Series Vol. 3. New Delhi: D. K. Printworld (P) Ltd. Sellato, B. 1989. Hornbill and Dragon. Jakarta: Elf Aquitaine Indonesie-Elf Aquitaine Malaysia. Setyawati, I. 1997. Knowledge and use of rice varieties in Apau Ping. In: Sorensen, K.W and B. Morris (ed.). People and Plants of Kayan Mentarang. London: WWF-Indonesia Programme. Sutlive V.H. 1978. The Iban of Sarawak. Illinois: AHM Publishing Corporation. Watson, G.A. 1984. Utility of Rice Cropping Strategies in Semuda Kecil Village, Central Kalimantan, Indonesia. In: Proceedings of the Workshop on Research Priorities in Tidal Swamp Rice, Banjarmasin 22-25 June 1981. Los Banos: International Rice Research Institute. Vayda, A.P. 1992. Report on Kayan Mentarang Consultancy, JulyAugust 1992. Report Kayan Mentarang Conservation Project. Jakarta: WWF and Direktorat Jenderal Pelestarian Alam dan Perlindungan Hutan. Widjaja, E.A., and T.C. Jessup. 1986. Short Description of Indigenous Rice from East Kalimantan, Indonesia. FAO/IBPGR Plant Genetic Resources Newsletter 67: 44-45.

BIODIVERSITAS Volume 10, Number 3, July 2009 Pages: 104-107

ISSN: 1412-033X (printed edition) ISSN: 2085-4722 (electronic) DOI: 10.13057/biodiv/d100209

The Occurrence of Hyperhydricity on Several Carnations (Dianthus carryophyllus L.) Cultivars during Low Temperature Storage KURNIAWAN BUDIARTO

Indonesian Ornamental Crops Research Institute (IOCRI), Cianjur 43253, West Java. Received: 5th December 2008. Accepted: 28th December 2008.

ABSTRACT The incident of hyperhydricity was a common problem in propagated carnation (Dianthus caryophyllus L.) during in vitro culture. Due to its possible relations with the decrease in phenotypic performance of plantlets, the observation on the occurrence of hyperhydricity was conducted on several in vitro conserved carnation cultivars. The research was conducted from July 2007 to August 2008 at The Indonesian Ornamental Crops Research Institute, Cianjur, West Java. A complete factorial experiment with 24 replications was designed to accomplish the combination of two factors. The first factor was six commercial carnation cultivars, namely light pink candy, malaga, opera, white candy, liberty and pink maladi, while the seconds dealt with type of conservation media, i.e. ½MS+DMSO 3%, ½MS+DMSO 3%+3% sucrose and control (½MS+3% sucrose). The results showed that the percentage of hyperhydric plantlet and plantlet viability after in vitro conservation were varied among carnation cultivars. Single treatment of sucrose had the least capacity in inducing plantlet resistance to low temperature conditions during in vitro conservation. Supplemental DMSO postponed the occurrence of hyperhydricity and with the existence of sucrose, higher plantlet viability were achieved. © 2009 Biodiversitas, Journal of Biological Diversity Key words: carnation (Dianthus caryophyllus L.), cultivar, hyperhydricity, plantlet viability, in vitro conservation.

INTRODUCTION Carnation or “anyelir” (Dianthus caryophyllus L.) is an important ornamental cut flower in the world due to their attractive and fragrance inflorescence. The plant belongs to family Caryophyllaceae and covers more than 8 genera with 2000 species. The endemic origins ranged from southern Russia to Alpine Greece and the Auvergne mountains of France (Jürgens et al., 2003). Various color and shape of flowers, leaves, flowering response and resistance to pests and disease in the existing cultivars have reflected the complex genetic construction resulted from tight breeding activities from the past. The trend for monoideotype cultivar in one hand has improved the preferred characters and incorporated in existing cultivars, while the undesirable traits are swept out (Ben-Yephet et al., 2005). The hastened genetic erosion on such plant has made a great emphasis of preserving genetic resources for future need. Native from temperate region, the plant had limited

 Corresponding address: Jl. Raya Pacet-Ciherang, PO. Box. 8 Sdl. Cianjur 43253, West Java Tel.: +62-263-512607, Fax.: +62-263-514138 e-mail: bud1arto@yahoo.com

life span in the tropics. The maintenance of huge genetic diversity for breeding to important characters in the field were then, very laborious and constrained with some technical problems such as decrease in viability after long clonal propagation and accompanying risks of pest and disease attacks, climatic perturbation and human error (Nugent et al., 1991). These condition were then, made in vitro conservation became an alternative way out in alleviating the limitation in vivo conservation. The incident of hyperhydricity, morphological, anatomical and physiological disorders of plantlet was a common problem in propagated carnation during in vitro culture. Hyperhydricity was characterized by stressed-glossiness plantlet appearance as results of deficiencies of cell wall edification and associated deficiency in organization of certain tissues such as the vascular bundles and the palisade tissue (Faguel et al., 2008), cuticle and wax deposition and stomata closure, reduced cell-to-cell adhesion resulting in breakability of the organs (Chen et al., 2006) and lack of chlorophyll accumulation and excess of water in intercellular spaces (Gaspar et al., 1995). Though most of reports referred to genotype dependent, some authors indicated the major causes of hyperhydricity was related with the characteristic of explants and the plantlet environment during in vitro

BUDIARTO – Hyperhidricity on carnation during low-temperature storage

culture, such as injury due to the prior dissection of the explants, typical fluxes of Ca and K ions, high relative humidity of the flask atmosphere, ammonium and cytokinine content of the media, and ethylene accumulation (Lai et al., 2005). The abnormal physiological process on hyperhydric plantlet almost all cases decreased phenotypic performance such the loss of organogenic potential which may correspond or be amore advanced form of diminished capacity to organize well structured stem and leaves formations and multiplication rate (Winarto et al., 2004). The in vitro conservation of carnation was successfully conducted with varying degree of viability among accessions using osmotic pressure method (Halmagyi and Deliu, 2007). Though the effects of DMSO (dimethylsulfoxide) as cell protectant on the incident of hyperhydricity has never been reported, the lengthened plantlet survives was presumably related with the action of such permeating agent on cell damage prevention against injurious effects of low temperature condition during in vitro conservation. Related with those facts, the observation on the occurrence of hyperhydricity was conducted on in vitro conserved carnation. In this paper, the affects DMSO conservation media on the abnormal hyperhydric incidence was investigated and further effects on carnation plantlet viability after certain duration of low temperature storage in induction medium was described.

MATERIALS AND METHODS The research was conducted in the Tissue Culture Laboratory at The Indonesian Ornamental Crops Research Institute, Cianjur, West Java from July 2006 to August 2007. A factorial complete experiment with 24 replications was designed to accomplish the combination of two factors. The first factor was six commercial carnation cultivars, namely light pink candy, malaga, opera, white candy, liberty and pink maladi. The second factor was type of modified conservation media, i.e. ½MS+DMSO 3%, ½MS+DMSO 3%+3% sucrose and control (½MS+3% sucrose). The rooted cuttings of carnation accessions were collected from commercial nurseries. The cuttings were then, replanted in 15 cm pot and maintained in protected glass house provided by twice a week foliar sprays of 2 g/L complete fertilizer. After 2 weeks, the plants were pinched and the new emerging lateral growths served for explants. The explants were disinfected using chemicals, then inoculated and subcultured into defined medium according to Budiarto et al. (2008) to obtain uniform plantlets. After three weeks subculture, 2 node-apical of plantlet was excised into treatment media and placed o into growth chamber on the temperature of 18-21 C. After three days, the plantlets were then preconditioned by lowering the temperature gradually

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(± 2 C every two days) until constant temperature of o 4 C. The viability of plantlets was evaluated and checked every two months during 12 months storage by subculturing the plantlet into induction medium. Prior to subculture, the culture flasks were placed into growth chamber with gradual temperature increase 0 up to 16-18 C (in six days). The observation was conducted on the percentage of hyperhydric plantlet, viability and other distinct phenomena related to the treatment being applied.

RESULTS AND DISCUSSION Conserved-plantlet of carnation cultivars during low temperature storage Type of media conservation gave significant influence on hyperhydric plantlet percentage and plantlet viability (Table 1). Similar phenomenon was observed on the carnation cultivars tested. Differences among cultivars were detected on all parameters evaluated during low temperature storage. However, no specific interrelation was found between the type of conservation media and carnation cultivar as far as these concerned. All the carnation cultivars tested showed hyperhydric symptom during in vitro conservation, though the values were varied among cultivars during 12 months low temperature storage (Table 1). The percentage of hyperhydric plantlets increased in line with the longer low temperature storage. Among the cultivars tested, cv. malaga showed the least hyperhydric plantlets in every 2 months observation up to 12 months in vitro conservation. In accordance with hyperhydric incidences, plantlet viability in induction medium was decreased with the lengthened storage duration. In every 2 months viability observation, cv. malaga still had the higher viable plantlet compared to other cultivars tested. Table 1. Percentage of hyperhydric plantlet and plantlet viability of carnation cultivars during low temperature storage. Carnation cultivars

Observation after……..months storage 2 4 6 8 10 12 Hyperhydric plantlet (%) a b b b bc b Light pink candy 1.6 23.6 45.3 74.3 92.5 100 a a a a a a Malaga 0 8.7 13.6 23.6 32.4 41.2 a bc b b b b Opera 2.5 28.3 42.5 79.3 88.6 93.3 a bc b bc bc b White candy 2.8 27.6 46.7 83.6 97.5 100 a c c c c b Liberty 4.8 33.1 58.7 87.6 100 100 a c c b bc b Pink maladi 7.3 36.7 62.8 78.3 93.2 100 Plantlet viability (%) a ab ab b ab a Light pink candy 100 78.4 54.3 40.7 17.7 4.2 a c d d d b Malaga 100 97.6 88.4 72.3 62.8 54.7 a a a a a a Opera 100 73.2 51.4 31.2 11.2 2.3 a a ab ab b a White candy 100 74.3 56.7 37.8 21.8 6.3 a b b bc bc a Liberty 100 87.3 62.3 44.2 27.3 2.1 a bc c c c a Pink maladi 100 91.7 76.4 56.4 33.4 3.7 *) Note: Values followed by different letters in the same column differ significantly at LSD 5%.

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B I O D I V E R S I T A S Vol. 10, No. 2, April 2009, pp. 104-107

Different performance among carnation cultivars on the percentage of hyperhydric plantlet and viability might refer to the different genetic constructions in each accession. These differences were also reflected on the ultrastructure microscopy of leaves. Chloroplast structure of normal plantlet of cv. malaga was merely unaffected though the hyperhydric symptoms were initially appeared after 4 months storage, while those on cv. pink maladi, the watery space was systematically increase on the incident of hyperhydricity (Figure 1). These phenomena were also reported on apple (Chakrabarty et al., 2005), coffea (Gatica-Arias et al., 2008), sun flower (Faguel et al., 2008) and potato (Turhan, 2004). The mode of gene action on the hyperhydricresistant genotype was still in debate up to this moment. Some authors stated that as generally most plant submitted to physical and chemical stresses, the plant possessed antioxidant defense against reactive oxygen species (ROS). These detrimental oxidative agents could attack unsaturated membrane lipids, nucleic acids, enzymes and other cellular structure (Cassi-Lit et al., 1997). In developing system against the ROS, the process involved various antioxidant compounds and a battery of antioxidant enzyme systems including catalase (CAT), superoxide dismutase (SOD), peroxidase (POD), glutathione peroxidase (GPX), glutathione S-transferase (GST) and the ascorbate glutathione cycle enzymes (Saher et al., 2004). On the study of normal Prunus avium plantlet, the activity of SOD was higher and functioned in disembarrassed the tissue of the accumulated superoxide ions by transforming them into H2O2. These H2O2 was itself eliminated through catalase involving ascorbate peroxidase, mono-and dehydro-ascorbate reductases and glutathione reductase, and ascorbate and glutathione. On the hyperhydric plantlet, the reduced-activity of such enzymes was almost always linked with DNA composition. It has been suggested then, the

decrease in DNA content affected in changes occurring at the cellular level as a result of stressinduced modifications of membrane composition at the abnormal morphology of hyperhydric leaves (Ochatt et al., 2002). Effects of media conservation on plantlet performance during in vitro conservation Percentage of hyperhydric plantlet during low temperature storage was varied among plantlets inoculated in media conservation. In general, the occurrence of hyperhydric plantlet was increasing in line with the duration of storage. The viability of the survival plantlet in induction medium at every 2 months observation during storage was also in accordance with the incident of hyperhydricity (Table 2). Table 2. Percentage of hyperhydric plantlet on different conservation media and respective plantlet viability in induction media after low temperature storage. Observation after……..months *) storage 2 4 6 8 10 12 Hyperhydric plantlet (%) a a b b b a ½MS+DMSO 3% 23.6 31.7 55.4 76.1 97.3 100 a a a a a a ½MS+DMSO 14.5 24.1 41.3 63.7 82.3 97.4 3%+3% sucrose b b c ½MS+3% sucrose 63.4 89.3 100 **) (control) Plantlet viability (%) b b b a a a ½MS+DMSO 3% 97.5 73.8 51.4 36.7 21.4 11.2 b c c b b b ½MS+DMSO 100 93.6 84.5 63.1 47.5 36.8 3%+3% sucrose a a a ½MS+3% sucrose 75.3 20.3 1.12 **) (control) *) Note: Values followed by different letters in the same **) column differ significantly at LSD 5%. no plantlet could be observed corresponding the death of plantlet after 8 months storage. Conservation media

CS CS

WS A

WS B

Figure 1. Chloroplast structure on palisade of (a) cv. malaga (normal) and (b) hyperhydric leaves tissue of cv. pink maladi after 4 months low temperature storage. The chloroplast cell and the watery space were pointed by white (CS) and yellow (WS) arrows respectively.

BUDIARTO – Hyperhidricity on carnation during low-temperature storage

The increase of hyperhydric plantlets in line with storage duration is evident that the cell biological system was disturbed in prolonged low temperature environment. During this condition, the cell failed to maintain its turgidity and viability and caused physiological disorder (Manoj et al., 2003). The more extreme impact on the plant corresponds to the early death of plantlet stored in ½ MS+3% sucrose and loss of viability for more than 50% of those stored in ½ MS+3% DMSO after 8 months storage (Table 2). The single treatment of sucrose or DMSO as cell protectant and preservative additives by inducing partial dehydration of cells was a failure in this study as also reported by Kartha et al. (1998). The ambiguous complex mechanism of sucrose and DMSO on increasing plantlet resistance to low temperature was observed when they were existing in combination. The higher plantlet viability in ½MS+DMSO 3%+3% sucrose than those stored in single treatment of DMSO or sucrose on every 2 months examination (Table 3) inferred that the mode of combined action of sucrose+DMSO was dependant on plantlet adaptation, though the percentage of hyperhydric plantlet were negligible with the single treatment of DMSO after 12 months. When the cell succeed to adjust osmotic balance between outer and inner by electrolyte accumulation and elicited membrane configuration induced by DMSO, the cell were then able to make use of sucrose to increase resistance to injurious dehydration (Zhao et al., 2005).

CONCLUCIONS Percentage of hyperhydric plantlet and plantlet viability after in vitro conservation were varied among carnation cultivars. Among the six cultivars tested, cv. malaga had the least hyperhydric plantlets and higher viability post to low temperature storage. Supplemental DMSO were able to postpone the occurrence of hyperhydricity and with the existence of sucrose, higher plantlet viability were achieved. Single treatment of sucrose, however, had the least capacity in inducing plantlet resistance to low temperature storage.

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OTHERS

The Occurance of Hyperhidricity on Several Carnations (Dianthus carryophyllus L.) Cultivars during Low Temperature Storage KURNIAWAN BUDIARTO

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Front cover: Dianthus carryophyllus L.

(PHOTO: KURNIAWAN BUDIARTO)

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