_________________________________________________________________ Agustini L, Wahyuno D, Santoso E. 2006. Keanekaragaman Jenis Jamur yang
Potensial dalam Pembentukan Gaharu dari Batang Aquilaria spp. (Biodiversity
of Potential Agarwood Inducer Fungi Taken from Aquilaria spp. Stems). Jurnal
Pusat Litbang Hutan dan Konservasi Alam 3(5): 555 -564
Terbentuknya gaharu diyakini sebagai respon pohon gaharu terhadap banyak
faktor, di antaranya fisiologis tanaman dan infeksi jamur.Sejumlah isolat jamur
3 yang berpotensi menginduksi gaharu telah diisolasi dari sampel kayu gaharu dari berbagai daerah. Penelitian ini ditujukan untuk mengetahui keanekaragaman jenis isolat yang berhasil dikoleksi.Sampel kayu diambil dari beberapa lokasi penanaman gaharu di Jawa, Sumatera, Kalimatan, dan Maluku. Kegiatan isolasi, pemurnian, dan perbanyakan dilakukan dengan menumbuhkan pada berbagai
media.Identifikasi dilakukan dengan mengamati ciri makroskopis dan mikroskopis
isolat yang dibiakkan pada media PDA dan BLA yang diinkubasi pada suhu
ruang dengan pencahayaan 300-400 lux selama 10-14 hari. Hasil identifikasi
menunjukkan bahwa biodiversitas isolat koleksi meliputi jenis Fusarium solani (Mart.) Appell and Walenw.,F. tricinctum (Corda) Sacc., F. sambucinum Fuckel, dan Cylindrocarponsp.
Kata kunci: Keanekaragaman, Fusarium, Cylindrocarpon, Makroskopis,
Mikroskopis
_________________________________________________________________ Ramadhani RC. 2006. Jaringan Pengakumulasi Resin Gaharu pada Aquilaria crassna. Skripsi Fakultas Matematika dan Ilmu Pengetahuan Alam (IPB)
Aquilaria crassna merupakan salah satu dari 17 spesies Aquilaria yang mampu menghasilkan resin gaharu. Pembentukan resin gaharu antara lain dapat disebabkan oleh invasi cendawan, pelukaan, dan perlakuan kimia. Resin yang dihasilkan oleh Aquilaria dilaporkan terakumulasi dalam jumlah besar di included phloem. Penelitian ini bertujuan mempelajari waktu terbentuknya included phloem pada akar, batang, dan daun A. crassna, serta mengamati peran jaringan lain dalam mengakumulasikan resin setelah tanaman diinduksi dengan Acremonium sp. Pembentukan dan perkembangan included phloem diamati pada tanaman umur 6 bulan dan 1 tahun, sedangkan pengamatan jaringan pengakumulasi resin gaharu diamati pada tanaman umur 1 dan 3 tahun. Irisan melintang seluruh organ tanaman umur 6 bulan, daun tanaman umur 1 dan 3 tahun dibuat dengan menggunakan mikrotom putar, sedangkan sayatan melintang akar dan batang tanaman umur 1 tahun serta cabang tanaman umur 3 tahun dibuat dengan menggunakan
mikrotom sorong. Tiga bulan setelah diinokulasi dengan Acremonium sp. jaringan
pengakumulasi resin gaharu diidentifikasi secara histokimia menggunakan larutan tembaga asetat [Cu(CH3COO)2·H2O], sedangkan kandungan pati diamati dengan
larutan I2KI. Included phloem sudah terbentuk pada akar dan batang A. crassna umur 6 bulan, sehingga induksi pembentukan gaharu dapat dilakukan pada umur tersebut. Included phloem tidak ditemukan pada daun tanaman A. crassna. Akumulasi resin pada jaringan tanaman yang terinfeksi oleh Acremonium sp. ditemukan di included phloem, unsur trakea xilem sekunder, dan parenkima jejari.
Kata Kunci: Jaringan, Resin Gaharu, Aquilaria crassna
_________________________________________________________________ Rusliani D. 2008. Pengaruh Asam Salisilat Terhadap Sifat Kayu Gaharu (Aquilaria crassna). Skripsi Fakultas Matematika dan Ilmu Pengetahuan Alam (IPB)
Asam salisilat merupakan lnolekul sinyal sekunder untuk pembentukan metabolit sekunder yang berfungsi sebagai senyawa pertahanan. Pengaruh asam salisilat terhadap pembentukan senyawa gaharu belum diketahui. lndikator pembentukan senyawa gaharu adalah perubahan warna dan adanya akumulasi terpenoid. Penelitian ini bertujuan mengetahui pengaruh pemberian asam salisilat terhadap perubahan warna kayu dan akumulasi terpenoid pada A. crassna. Cabang pohon A.crassna yang berumur 8 tahun (berdiameter 1cm) dibuang kulit dan kambiumnya sepanjang 2 cm kemudian dioleskan asam salisilat dengan konsentrasi lOOn1M (SA loo), 200mM (SA 200), 300mM (SA 300), 4001nM (SA 400), 700mM (SA 700) dan lOOOmM (SA 1000). Tingkat warna kayu dan besarnya zona perubahan warna kayu, akumulasi terpenoid (uji histokiinia dan Liebemian-Burchard) dan tingkat wangi diamati pada 5, 10,25 dan 50 hari setelah pemberiana asam salisilat (hsa). Pemberian asam salisilat menyebabkan perubahan warna kayu dari putih menjadi
putih kecoklatan yang disertai munculnya gejala fisiologis,seperti klorosis (SA 400
pada 50 hsa dan SA 700 pada 5 hsa), batang rapuh dan kematian cabang (SA 700 pada 25 hsa dan SA 1000 pada 50 hsa). Kandungan terpenoid inenyebabkan perubahan warna kayu. Akumulasi terpenoid ditemukan pada parenkima jejari, included phloem, unsur trakea xilem dan empulur kecuali pada kayu yang inendapat perlakuan SA 700 dan SA 1000, akumulasi terpenoid hanya terdapat di parenkima jejari dan unsur trakea xilem. Akurnulasi lemak ditemukan pada parenkima jejari, includedphloem, dan unsur hitkea xilem. Sedangkan akumulasi pati ditemukan pada 5-10 hsa di parenkima jejari dan inclsided phloem. Warna kuning pada uji Lieberman Burchard menunjukkan adanya kandungan sterol. Pemberian asam salisilat dapat menginduksi pembentukan senyawa terpenoid tapi tidak dapat menginduksi wangi gaharu.
Kata Kunci: Asam Salisilat, Gaharu, Aquilaria crassna, Sifat Kayu
Eurlings MCM, Heuveling van BeekH, Gravendeel B. 2010. Polymorphic
Microsatellites for Forensic Identification of Agarwood (Aquilaria crassna).
Forensic Science International 197 (1): 30-34
Tropical agarwood (Aquilaria) is in danger of extinction in the wild due to illegal logging. Its resin (Gaharu) is used for the production of highly valued incense throughout Asia. We have isolated and characterized microsatellite loci of Aquilaria crassna to detect the geographic origin of agarwood for forensic applications using
a modified enrichment procedure based on the capture of repetitive sequences from restricted genomic DNA. We assessed the polymorphisms of five microsatellites amplified from fresh leaves of 22 trees from seven plantations in Vietnam and Thailand and dried leaves of a herbarium specimen of one wild tree. Cross specificity of these markers was confirmed on two related Aquilaria species occurring in China
and Vietnam and one microsatellite locus was successfully amplified from wood
and incense samples. Four of the loci were polymorphic and the number of alleles ranged from 3 to 15. The loci characterized here can provide a starting point for
forensic identification of traded material and certification of sustainably produced
agarwood.
Keywords: Gaharu, Microsatellites, Thymelaeaceae, Tropical forest products,
Wildlife forensics
_________________________________________________________________ Kumeta Y, Ito M. 2010. Characterization of [delta]-Guaiene Synthases
from Cultured Cells of Aquilaria, Responsible for the Formation of the
Sesquiterpenes in Agarwood. Plant Physiology 154 (4): 1998-2007
The resinous portions of Aquilaria plants, called agarwood, have been used as medicines and incenses. Agarwood contains a great variety of sesquiterpenes, and a study using cultured cells of Aquilaria showed the production of sesquiterpenes
(α-guaiene, α-humulene, and δ-guaiene) to be induced by treatment with methyl
jasmonate (MJ). In this study, the accumulation and production of sesquiterpenes
were quantified. The amounts accumulated and produced reached a maximum at 12 h, and the most abundant product was α-humulene at 6 h and δ-guaiene after 12 h. However, a headspace analysis of the cells revealed that α-humulene
is likely to be volatilized; so overall, the most abundant sesquiterpene in the cells
was δ-guaiene. A cDNA library from RNA isolated from MJ-treated cells was screened using PCR methodologies to isolate five clones with very similar amino
acid sequences. These clones were expressed in Escherichia coli, and enzymatic reactions using farnesyl pyrophosphate revealed that three of the clones yielded the same compounds as extracted from MJ-treated cells, the major product being
δ-guaiene. These genes and their encoded enzymes are the first sesquiterpene
synthases yielding guaiene-type sesquiterpenes as their major products to be reported. Expression of a fourth terpene synthase gene in bacteria resulted in
the accumulation of the protein in insoluble forms. Site-directed mutagenesis of the inactive clone and three-dimensional homology modeling suggested that the structure of the N-terminal domain was important in facilitating proper folding of the protein to form a catalytically active structure.
Keywords: Aquilaria, Agarwood, Sesquiterpenes, Guaiane, Ligases
_________________________________________________________________ Murtaip. 2010. Induction of Agarwood Formation by Combination of Acremonium and Chemical Treatments. Skripsi Fakultas Matematika dan Ilmu Pengetahuan Alam (IPB)
Agarwood is an aromatic resin produced by Aquilaria spp. The resin was formed as a response to fungal infection, mechanical damage or chemical treatment. This research was aiming at evaluation of the effectivity of single induction agent (salicylic acid (A), methyl jasmonate (M), Acremonium (F)) and in comparison to combination of induction agents. Trunks of Aquilaria crassna (±10 years old) was induced by either a single induction agents (A, M or F) or its combination (AF, AM, MF or AMF). Acremonium was given as pellets, while A and M were given at 100
mM and 750 mM, respectively. The tree was first drilled (4 mm diam. and one third
diameter of the stem), with 20 cm distance between holes. The trunk was then injected with A, subsequently with M and inoculated with F, with a week interval of teratments. Agarwood quality criteria such as wood discoloration, fragrance level, and also terpenoid accumulation were observed monthly for four months periode. The results indicated that intensity of wood discoloration of all treatments ranges from brownish white to dark brown with AMF combination gave highest intensity. Fragrance did not induce by all treatments with AMF combination given highest mean score, but on the base at percentage of point induction, MF and F were more effective to induce fragrance. All of treatments induced terpenoids formation. AM and F treatments induced high triterpenoid accumulation with concentration 268.25 ppm. Wood discoloration was not correlated with fragrance level, but it was correlated with terpenoid accumulation. AMF stimulated wood discoloration and its fragrance level was better than the other treatments. However, only MF and F were
effective to induce spesific agarwood fragrance.
Keywords: Aquilaria crassna, Acremonium, Salycilic acid, Methyl jasmonate,
discoloration, terpenoid.
Nimnoi P, Pongsilp N, Lumyong S. 2010. Endophytic Actinomycetes Isolated
from Aquilaria crassna Pierre ex Lec and Screening of Plant Growth Promoters
Production. World Journal of Microbiology and Biotechnology 26 (2):193-203
A total of 10 endophytic actinomycete strains were successfully isolated from healthy shoots and roots of Aquilaria crassna Pierre ex Lec (eaglewood). Analysis of 16S rDNA sequencing of those isolates showed that they belong to members of the genera Streptomyces (2 isolates), Nonomuraea (1 isolate), Actinomadura (1 isolate), Pseudonocardia (1 isolate) and Nocardia (3 isolates). The remaining 2
isolates were unidentified. All of isolates produced the amount of indole-3-acetic acid (IAA) and ammonia ranging between 9.85 ± 0.31 to 15.14 ± 0.22 μg ml^sup -1^ and 2 to 60 mg ml^sup -1^, respectively. Among 10 isolates tested, the amount
of hydroxamate-type siderophore produced by 2 isolates was undetectable. While the remaining 8 isolates produced the amount of hydroxamate-type ranging
between 3.21 ± 0.12 and 39.30 ± 0.40 μg ml^sup -1^. Also, catechols-type siderophore produced by 9 isolates was undetectable. Actinomadura glauciflava is only one isolate that produced catechols-type 4.12 ± 0.90 μg ml^sup -1^. In
addition, 10 endophytic actinomycetes showed protease activity ranging from
undetectable to 8.16 ± 0.15 unit ml^sup -1^. Genetic relatedness amongst these isolates was determined base on Random amplified polymorphic DNA (RAPD) and
Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC
PCR). Both methodologies generated specific patterns corresponding to particular genotypes. RAPD fingerprinting proved to be slightly more discriminatory than ERIC PCR. This study is the first published report that actinomycetes can be isolated as endophytes within this plant. It is also the first published report that endophytic
actinomycetes are capable of producing IAA and siderophores.
Keywords: Endophytic actinomycetes, Idole-3-acetic acid, Siderophores,
Enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC
PCR), Random amplified polymorphic DNA (RAPD)
_________________________________________________________________ Wilarso SBR, Santoso E, Wahyudi A. 2010. Identifikasi Jenis-jenis Fungi
yang Potensial terhadap Pembentukan Gaharu dari Batang Aquilaria spp.
(Identification the Potential Types of Fungi on Establishment Agarwood Stem
of Aquilaria spp.). Jurnal Sivikultur Tropika 1 (01): 1 – 5
One of tree genus that becomes the main source of agarwood producer is Aquilaria. The formation of agarwood was believed as response of agarwood trees to many factors, likes plant physiological and pathogenic fungi infection. A number of isolates that have the potential to induce agarwood has been isolated from wood samples from different areas such as Bangka, Bogor, Bohorok, Gorontalo, East Kalimantan, Lampung, Mentawai, Papua, and Sukabumi. This study aimed to obtain information on the types of fungi isolated from stem-forming agarwood Aquilaria
spp. that successful collection of Microbiology Laboratory of Forestry Research in
Bogor. Identification was done by observing morphological features from cultured isolates on PDA and BLA. The results of identification of isolates collection include
species of Fusarium solani (Mart.), Appell and Walenw, F. tricinctum (corda) Sacc, F sambucinum Fuckel, and F. Moniliformae.
Keywords: Identification, fungi, Fusarium, Aquilaria spp.
_________________________________________________________________ Hodijah RS. 2011. Cendawan dan Bahan Kimiawi sebagai Formula Inokulan
Penginduksi Gaharu. Skripsi Departemen Biologi Fakultas Matematika dan Ilmu
Pengetahuan Alam (IPB)
Penelitian ini dilakukan untuk mengetahui pengaruh asam salisilat 0,1 mM dan metil jasmonat 0,75 mM terhadap pertumbuhan Acremonium sp. (IPBCC 08.566, IPBCC 07.525) dan Fusarium sp. (IPBCC 08.568, IPBCC 08.569 dan IPBCC 08.570) pada media laboratorium dan formula inokulan berbentuk pelet. Acremonium sp. atau Fusarium sp. (diam. 5 mm) ditumbuhkan pada media PDA dan dipasangkan dengan lempeng kertas saring (diam. 5 mm) yang mengandung 1 mL asam salisilat 0,1 mM atau metil jasmonat 0,75 mM. Pengamatan dilakukan terhadap diameter koloni dan jumlah konidia setiap dua hari sampai koloni memenuhi cawan. Senyawa kimia yang tidak berpengaruh terhadap pertumbuhan diteliti lebih lanjut sebagai komponen formulasi pelet dan pelet diuji viabilitasnya. Uji viabilitas dilakukan dengan menumbuhkan cendawan berupa pelet pada media PDA. Asam salisilat 0,1 mM atau metil jasmonat 0,75 mM tidak berpengaruh terhadap pertumbuhan miselium dan produksi konidia pada semua isolat. Namun asam salisilat 0,1 mM merangsang pembentukan konidia Fusarium IPBCC 08.568 (sebesar 1,005x produksi konidia kontrol). Secara umum Acremonium sp. maupun Fusarium sp. pada pelet dengan penambahan asam salisilat 0,1 mM atau metil jasmonat 0,75 mM dan 750 mM memiliki viabilitas sebesar 85-95,8% dan tidak berbeda nyata dari kontrol yaitu sebesar 90-96,6%. Sedangkan semua cendawan pada pelet yang diberi asam salisilat 100 mM tidak tumbuh sehingga dianggap tidak viabel.
Kata kunci: Cendawan, Inokulan, Gaharu
_________________________________________________________________ Jin-long C, Shun-xing G, Pei-gen X. 2011. Antitumor and Antimicrobial Activities
of Endophytic Fungi from Medicinal Parts of Aquilaria sinensis. Journal of
Zhejiang University - Science B 12(5): 385–392
The purpose of this study was to isolate and characterize endophytic fungi from the stem tissue which can produce fragrant ingredients in Aquilaria sinensis (also called agarwood) to determine their antitumor and antimicrobial activities. Twenty-eight fungal endophytes were isolated fromagarwood by strict sterile
sample preparation and were classified into 14 genera and 4 taxonomic classes
(Sordariomycetes, Dothideomycetes, Saccharomycetes, and Zygomycetes) based
on molecular identification. Of the 28 isolates, 13 (46.4%) showed antimicrobial
activity against at least one of the test strains by the agar well diffusion method, and
23 isolates (82.1%) displayed antitumor activity against at least one of five cancer
cell lines by 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The diameters of inhibition zones of YNAS07, YNAS14, HNAS04, HNAS05, HNAS08, and HNAS11 were equal to or higher than 14.0 mm against Staphylococcus aureus, Escherichia coli, Bacillus subtilis, B. subtilis, Aspergillus fumigatus, and B. subtilis, respectively. The inhibition rates of YNAS06, YNAS08, and HNAS06 were not less than 60% to 293-T, 293-T, and SKVO3 cells, respectively. These results suggest that the endophytic fungi associated with agarwood will provide us with not only useful micro-ecological information, but also potential antimicrobial and antitumor agents.
Keywords: Agar diffusion method, Agarwood, Antimicrobial bioactivity, Antitumor
bioactivity, Endophytic fungi.
_________________________________________________________________ Lia PA. 2011. Studi Interaksi Fusarium sp. dengan Pohon Gaharu (Aquilaria
sp.) Menggunakan Pendekatan Sitologi. Skripsi Bogor Agricultural University
(IPB)
The research is aiming to study the pathogenesis mechanism of Fusarium sp. IPBCC. 08.569 on Aquilaria sp. seedling in respect to the respons of the host towards Fusarium sp. IPBCC. 08.569 infection. Penetration mechanism was determined by observing the appresorium formation on the bark surface in vitro, the potency to produce lignocellulase and toxin. While colonization was studied by histopathological methods using both staining process and SEM. The respons of the tree towards infection was observed at either cellular by histochemestry or organ level using scoring system. Fusarium sp. IPBCC. 08.569 showed no appresorium formation but had lignocellulolytic activity. Hypersensitivity reaction of tobacco leaf towards crude extract of extracellular substance indicated that Fusarium sp. IPBCC. 08.569 produce toxin. Direct penetration into the bark was not found, therefore penetration
had to be through wounded area. While modification of hyphae was observed in the
living cells of the wood tissue (parenchyma rays, included phloem, and pith) these cells might contain terpenoid as conversion of starch. These cellular changes might be observed as discolored wood and the wood became scented. Discolored wood was extended outside the colonization area, indicated that the dark substance might be part of hypersensitive respons to delimit the infection. Using scoring system, after inoculation Fusarium sp. IPBCC. 08.569 can be stated as wound infection fungi with limited colonization in living tissue and induce terpenoid formation in certain tissue.
Rahayu G, Santoso E, Wulandari E. 2011. Effectivity and Interaction Between
Acremonium sp. and Fusarium sp. in Formation of Gaharu Clump in Aquilaria microcarpa. Indonesia’s Work Programme for 2011 ITTO PD425/06 Rev.1 (I) R&D Centre for Forest Conservation and Rehabilitation pp 47- 57
Aquilaria microcarpa is one of the trees that produce gaharu. Gaharu is formed as a response to a fungus infection. Acremonium sp. and Fusarium sp. were the fungus which often used to induce clump formation. Both these fungus were often isolated from one single clump symptom. Interaction between both fungus in clump formation was unknown. Therefore the ability of Acremonium sp. and Fusarium sp. and their interaction in clump formation were to be studied. Tree trunks of A. microcarpa were drilled and then inoculant 1 (A= Acremonium sp. or F= Fusarium sp.) was inserted into a sequence of holes and followed by inoculant 2 (F=Fusarium sp. or A=Acremonium sp). into another sequence of holes with 1 week interval on the same tree trunk. Before the inoculant Acremonium was inserted into the holes, the holes were treated with 2% sugar solution. Range between a sequence of holes of inoculant 1 and inoculant 2 was 15 cm. All treatments consisted treatment with single inoculant AA and FF, with double inoculant AF and FA, without inoculant (only drilled =B, drilled and treated with sugar=G), and negative control (K). Range between holes of a pair of treatments was 30 cm. Every treatment was made in 3 different trees. Effectivity and interaction between inoculant were determined by length, width of color-change zone on wood, color level, fragrant level, and precentage of fragrant induction point, and terpenoid compound accumulation. Wood color change level and fragrant level were determined by Liebermann- Burchard method. Observation was carried every month for 4 months. Generally, every treatment caused color change on wood and stimulated wood’s fragrant change. Sugar solution caused the symptom of gaharu clump formation supressed. Acremonium and Fusarium were relatively more effective in stimulating the gaharu clump formation rather than holes- making or sugar solution treatment, especially in inducing fragrance. Double inoculant treatments, especially AF was more effective in inducing fragrance formation than FA and single inoculant. On the other side, inoculant FA was better at other parameters. With 1 week interval, inoculant 1 did not raise resistence to inoculant 2, likewise, inoculant 2 did not seem to affect inoculant 1. Terpenoid compound which is classified into triterpenoid was detected in all double treatments and single treatment F. In other treatment, sterol compound was found. The concentration of both compounds were lower than those found in nature gaharu.
Keywords: Terpenoid compound, Aquilaria microcarpa, Acremonium, Fusarium
Turjaman M, Santoso E, Sitepu IR, Osaki M, Keitaro T. 2011. Application of
Arbuscular Mycorrhizal Fungi in Four Speciesof Aquilaria. Indonesia’s Work
Programme for 2011 ITTO PD425/06 Rev.1 (I) R&D Centre for Forest Conservation and Rehabilitation pp 79 – 87
The scarcity of natural gaharu (agarwood) production is due to excessive exploitation in Indonesian tropical natural forest. The sustainability of mother trees which produce gaharu is disturbed due to many activities of felling the trees, so that there is a threat of extinction, particularly for species of Aquilaria. Afterwards, the availability of natural regeneration seeds which produce gaharu, become also limited. The main problems addressed in this research is the slow growth of Aquilaria, either in
the nursery or in the field, due to acid soil condition and nutrient deficiency. The use
of arbuscular mycorrhizal (AM) fungi is possible to help the initial growth of Aquilaria species in the acid soils. The objective of this research was determining the effect of several AM fungi species on Aquilaria species, either in the nursery or in the field. Species of Aquilaria used in this reserach were Aquilaria malaccensis, A. crassna, A. microcarpa and A. beccariana. Species of AM fungi being used in this study were Entrophospora sp., Gigaspora decipiens, Glomus clarum, Glomus sp. ZEA, and Glomus sp. ACA. This research used completely randomized experimental design with 30 replications. Parameters observed in this research were AM fungi