Journal Of Agrobiotechnology (Journal of UniSZA - Universiti Sultan Zainal Abidin)
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Internal Transcribed Spacer (ITS) as the Molecular Marker for Identification of Dioscorea hispida Dennst. (Dioscoreaceae)
Get PDFDioscorea hispida is a source of food and also has important medicinal values. However, there are difficulties in identifying and recognizing D. hispida based on morphological characteristics. D. hispida collected from Besut District in the state of Terengganu, Malaysia, were subjected to PCR amplification with ITS1 and ITS2 (universal primers) but no amplified product was obtained. Therefore, a n ew set of primers was designed based on a specific signature sequence for Dioscorea spp. in the ITS region named as Dios_003 (5’-AAA CGG CTA CCA CAT CCA AG-3’) and Dios_004 (5’-AGA CAA ATC GCT CCA CCA AC-3’). A specific band of 900 bp was detected for all accession numbers of D. hispida. Sequencing of the amplified ITS region was carried out and alignment of the sequences demonstrated that the ITS region amplified was identical in all the D. hispida accessions and was highly conserved among other Dioscorea spp. that were analyzed, i.e. D. japonica, D. alata, D. elephantipes, D. communis, D. caucasia, D. sylvatica and D. polystachya. Therefore, the new set of internal transcribed spacer (ITS) marker is an effective marker to identify D. hispida. Keywords: Dioscorea hispida, Internal Transcribed Spacers (ITS), molecular marker ABSTRAK Dioscorea hispida adalah salah satu sumber makanan dan ia juga penting dalam nilai perubatan. Bagaimanapun, terdapat masalah dalam menentukan dan mengenalpasti D. hispida melalui ciri-ciri morfologi. D. hispida yang diperoleh daripada daerah Besut, Terengganu telah diamplifikasikan menggunakan PCR dengan pencetus universal ITS1 dan ITS2 tetapi tiada amplifikasi yang diperoleh. Oleh yang demikian, satu set pencetus baru telah direka berdasarkan jujukan tandatangan spesifik D. hispida iaitu Dios_003 (5’-AAA CGG CTA CCA CAT CCA AG-3’) dan Dios_004 (5’-AGA CAA ATC GCT CCA CCA AC-3’). Satu jalur spesifik bersaiz 900 pb telah dikesan dalam kesemua aksesi D. hispida. Penjujukan kawasan ITS yang dikuatkan telah dilaksanakan dan penjajaran kesemua jujukan tersebut menunjukkan bahawa kawasan ITS yang dikuatkan adalah sama dalam kesemua aksesi D. hispida dan sangat terabadi antara Dioscorea spp. yang lain yang telah dianalisis, iaitu D. japonica, D. alata, D. elephantipes, D. communis, D. caucasia, D. sylvatica dan D. polystachya. Kata kunci: Dioscorea hispida, Internal Transcribed Spacers (ITS), penanda molekul
Enzymatic Synthesis of Structured Lipids through Interesterification of Palm Stearin (POs) IV 14, Cottonseed Oil (CSO) and Palm Olein (POo)
Get PDFABSTRACTStructured lipids were produced from ternary blending of palm stearin (POs) with iodine value (IV=14), cottonseed oil (CSO) and palm olein (POo) through enzymatic interesterification (IE). Eighteen ternary blends of POs:CSO:POo that were produced from Mixture Procedure (D-Optimal) in Design Expert®version 6 were enzymatically interesterified using TLIM lipase (10%, wt/wt substrate) for 24 hr at 55°C. Blends without enzyme were used as comparison, while commercial vanaspati of Dalda brand was used as a control. Physico-chemical characteristics such as solid fat content (SFC), iodine value (IV), slip melting point (SMP) and melting/crystallization behavior of all blends and commercial samples were studied (Stage one). Blend samples with SFC similar to Dalda were chosen and their triacylglycerol (TAG), fatty acids compositions and peroxide value (PV) were determined. Results from the study showed that IV of blends without enzyme were in the range of 30.4 – 80.0, while IE blends were 33.5 – 74.4. The SMP of blends without enzyme were in the range of 50 – 59°C and for IE blends, the value was decreased in the range of 44 – 53°C. At stage two, the SFC profile for blends no. 3 (POs:CSO:POo; 20:23.33:56.67) and no. 15 (POs:CSO:POo; 20:10:70) with enzyme and without enzyme, had profiles quite similar to Dalda. SFC values for blend no. 3 with and without enzyme, at 20°C were 23.53% and 17.99%, respectively. For blend no. 15, SFC with and without enzyme were 22.78% and 23.19%, respectively at 20°C. Fatty acid composition for both blends showed no significant difference.Keywords: Enzymatic synthesis, interesterification, cottonseed oil, Dalda (vanaspati) ABSTRAKLipid berstruktur dihasilkan dengan menggunakan adunan tigaan stearin sawit (POs) dengan nilai iodin (IV=14), minyak biji kapas (CSO) dan minyak olein sawit (POo) melalui proses interesterifikasi berenzim (IE). Sebanyak 18 adunan tigaan POs:CSO:POo yang terhasil dari Proses Pengadunan (D-Optimal) di dalam Design-Expert®versi 6 telah dilakukan interesterifikasi berenzim dengan menggunakan lipase TLIM (10%, b/b substrat) selama 24 jam pada suhu 55°C. Adunan tanpa enzim telah digunakan sebagai perbandingan, manakala sampel vanaspati dagangan jenama Dalda telah digunakan sebagai kawalan. Kajian ciri-ciri fizik-kimia seperti kandungan lemak pepejal (SFC), nilai iodin (IV), takat lebur gelincir (SMP) dan kelakuan peleburan/penghabluran telah dilakukan ke atas kesemua adunan dan sampel dagangan (peringkat pertama). Sampel adunan yang mempunyai SFC menyamai Dalda dipilih dan ditentukan komposisi triasilgliserol (TAG), asid lemak dan nilai peroksida (PV). Hasil daripada kajian mendapati nilai IV untuk adunan tanpa enzim ialah antara 30.4 – 80.0 dan adunan IE ialah antara 33.5 – 74.4. Nilai SMP bagi adunan tanpa enzim ialah 50 – 59°C dan nilai SMP bagi adunan IE menurun iaitu 44 – 53°C. Pada peringkat kedua, profil KLP untuk adunan no. 3 (POs:CSO:POo; 20:23.33:56.67) dan no. 15 (POs:CSO:POo; 20:10:70) berenzim dan tanpa enzim, mempunyai profil yang hampir sama dengan Dalda. Nilai KLP bagi adunan no. 3 berenzim dan tanpa enzim, pada suhu 20 °C ialah 23.53% dan 17.99%, secara berturutan. Bagi adunan no. 15, nilai KLP berenzim dan tanpa enzim ialah 22.78% dan 23.19%, secara berturutan pada suhu 20°C. Komposisi asid lemak bagi kedua-dua nisbah tidak menunjukkan perbezaan yang ketara.Kata kunci: Sintesis berenzim, interesterifikasi, minyak biji kapas, Dalda (vanaspati
Effects of Culture Age on Conidia Production, Viability and Pathogenicity of Dactylaria higginsii
Get PDF Effects of culture age on conidia production, viability and pathogenicity of Dactylaria higginsii, a potential bioherbicide for controlling Cyperus rotundus (purple nutsedge) were investigated both in the laboratory and the greenhouse. This fungus was capable of producing conidia after five days of culturing on potato dextrose agar (PDA), with the highest conidia production obtained from the 15 day-old culture compared to other culture ages. Conidia harvested from 15 days of culturing on PDA, produced higher viability and were more pathogenic to purple nutsedge compared to conidia harvested before or after 15 days of culturing. The infection rate of conidia harvested from the 15 day-old cultures was faster (rL0.40 logit/day) compared to the infection rate of conidia harvested from other culture ages. Keywords: Mycoherbicide, purple nutsedge, Dactylaria higginsii, harvesting age Kesan usia kultur kulat ke atas penghasilan konidia, kemandirian dan kepatogenan Dactylaria higginsii, kulat yang berpotensi sebagai bioherbisid untuk mengawal Cyperus rotundus (rumput halia hitam) telah dikaji pada keadaaan makmal dan rumah hijau. Dactylaria higginsii berupaya menghasilkan konidia selepas lima hari setelah dikulturkan di atas agar dekstosa ubi kentang (PDA), dengan penghasilan konidia tertinggi pada hari yang ke 15, dengan kebolehidupan tertinggi dan lebih patogen kepada C. rotundus berbanding konidia pada usia sebelum hari yang ke 15. Kadar jangkitan konidia yang berusia 15 hari juga lebih cepat (rL0.40 logit/hari) berbanding kadar jangkitan konidia pada hari-hari yang lain. Kata kunci: Mikoherbisid, rumput halia hitam, Dactylaria higginsii, umur penuaia
Potential of Exserohilum monoceras as a Bioherbicide for Controlling Echinochloa crus-galli (Rumput Sambau)
Get PDFThe fungus isolated from diseased Echinochloa crus-galli was identified as Exserohilum monoceras, based on the conidial morphology, germination characteristics and hilum morphology. The objectives of this study were to screen and isolate fungal pathogen of E. monoceras from infected E. crus-galli as well as to determine the pathogenicity of E. monoceras and the optimum conditions for growth and sporulation of E. monoceras. In repeated trials, the pathogenicity of this fungus to its host was confirmed by Koch’s postulate. The pathogen caused a high level of disease on E. crus-galli where symptoms appeared within 24 h of inoculation. It induced eyespot lesions typical of infection by Exserohilum spp. and induced a severe ‘burnt’ appearance on the foliage of E. crus-galli. The leaf tissues in the vicinity of heavy infestation were completely killed. The disease did not develop on non-inoculated plants, which was indicative of localized infection and lack of significant secondary disease cycles. The laboratory study indicated that better growth and appressoria production can be attained when the fungus is exposed to temperature in the range of 25 to 30 oC. This study also indicated that Maxigreen®, which is an adjuvant, when mixed with sunflower in the ratio of 0:5:7 is ideal for formulating the conidia. This research has illustrated that solid media such as agar is an excellent growth media, but spore production has been influenced by the nutrient content of the media. The best medium for sporulation was half strength V8 juice agar. Keywords: Exserohilum monoceras, Echinochloa crus-galli, Koch’s postulate, infection, conidia, sporulation Kulat yang dipencilkan dari Echinochloa crus-galli yang berpenyakit telah dikenalpasti sebagai Exserohilum monoceras berdasarkan morfologi konidia, ciri-ciri percambahan dan morfologi hilum. Objektif kajian ini adalah untuk mengenalpasti kulat patogen iaitu E. monoceras daripada E. crus-galli yang berpenyakit dan mengenalpasti kepatogenan E. monoceras dan kadar yang optimum untuk pertumbuhan dan sporulasi E. monoceras. Dalam percubaan ulangan, kepatogenan kulat ini pada perumah adalah terbukti dengan postulat Koch. Patogen ini menyebabkan penyakit pada kadar yang tinggi pada E. crus-galli dan simptom akan kelihatan dalam masa 24 jam. Selepas inokulasi, kelihatan lesion bintik mata yang merupakan simptom tipikal jangkitan oleh Exserohilum spp. Turut kelihatan adalah keadaan ‘terbakar’ pada helaian daun E. crus-galli. Tisu daun pada kadar jangkitan yang teruk akan mati sepenuhnya. Jangkitan tidak berlaku pada pokok yang tidak diinokulasi, membuktikan jangkitan setempat dan tiada jangkitan kitaran kedua. Kajian makmal menunjukkan pertumbuhan dan penghasilan apresoria yang sesuai adalah pada suhu sekitar 25 hingga 30 °C. Kajian ini juga menunjukkan Maxigreen®, iaitu sejenis agen pelembap, adalah merupakan formulasi terbaik untuk penghasilan konidia apabila dicampurkan dengan minyak bunga matahari pada kadar 0:5:7. Kajian ini turut membuktikan bahawa media pepejal seperti agar adalah media pertumbuhan terbaik, tetapi penghasilan spora adalah dipengaruhi oleh kandungan nutrien di dalam media dan media terbaik untuk sporulasi adalah agar jus V8 separuh kekuatan. Kata kunci: Exserohilum monoceras, Echinochloa crus-galli, postulat Koch, jangkitan, konidia, sporulas
PCR-RFLP of Ficus deltoidea Jack (Moraceae)
Get PDFPrevious taxonomic determination of Ficus deltoidea Jack was mainly based on narrative morphology. However, the classification and relationship of some F. deltoidea varieties were disputable. In this study, four universal primers were used to amplify chloroplast DNA and were digested with eight restriction enzymes to obtain intravarietal variation among Ficus deltoidea var. deltoidea, Ficus deltoidea var. trengganuensis, Ficus deltoidea var. intermedia, Ficus deltoidea var. kunstleri, Ficus deltoidea var. angustifolia, and Ficus deltoidea var. bilobata. Pre-screening of six cpDNA primers revealed that the rbcL and rpoB amplified fragments contained restriction sites for HindIII and RsaI. However, no polymorphism was observed by using this method. Keywords: Ficus deltoidea Jack, chloroplast DNA, PCR-RFLP ABSTRAK Penentuan taksonomi bagi Ficus deltoidea Jack secara asasnya adalah berdasarkan kepada kaedah morfologi. Walaubagaimanapun, pengkelasan dan perhubungan antara beberapa varieti Ficus deltoidea telah dipertikaikan. Dalam kajian ini, sebanyak empat pencetus universal telah dipilih dan digunakan untuk mengamplifikasi kawasan DNA kloroplas dan kemudian dicernakan oleh lapan enzim penyekat untuk mendapatkan variasi intravarieti di antara Ficus deltoidea var. deltoidea, Ficus deltoidea var. trengganuensis, Ficus deltoidea var. intermedia, Ficus deltoidea var. kunstleri, Ficus deltoidea var. angustifolia dan Ficus deltoidea var. bilobata. Kawasan cpDNA, telah diamplifikasikan dengan menggunakan enam pencetus cpDNA tetapi hanya pencetus rbcL dan rpoB mempunyai tapak penyekatan untuk HindIII dan RsaI. Namun, tiada polimorfisma yang dapat dicerap dengan menggunakan kaedah ini. Kata kunci: Ficus deltoidea Jack, DNA kloroplas, PCR-RFL
Application of the Protide Strategy to Some Novel Nucleoside Analogues
Get PDFThe majority of HIV-Herpes nucleoside analogues share a common mode of action, which is conversion via three successive phosphorylations, to their bioactive 5’-triphosphate forms by cellular or viral induced kinases. The protide strategy aims to deliver the monophosphate form of the parent nucleoside, in order to by-pass the first phosphorylation step and thus, the need for kinase-mediated activation. Indeed, a number of protide analogues have retained full anti-viral activity in kinase-deficient cells, suggesting that the protides effectively deliver the 5’-monophosphate forms intracellularly. In this paper the phosphoramidate derivatives of the thymine nucleoside analogues, as well as the 3'-amino-AZT derivative were synthesized and assayed for their antiviral activities. These derivatives exhibited poor activities, when compared to the parent nucleoside, indicating a lack of successful nucleotide delivery. Nevertheless, the formation of by-products during the synthesis of phosphoramidates via the phosphorochloridate strategy was overcome by controlling the conditions of the reaction and especially by choosing a selective coupling reagent, such as tBuMgCl or N methyl imidazole to avoid any N-1 or 3’ -phosphorylation. It is also clear from the results that phosphoramidates were sensitive to harsh conditions and reagents such as hydrogenation. The addition of the phosphochloridate should always be left to the last stage
Leaf Surface Characteristics of Selected Malaysian Weed Species of Oil Palm
Get PDFLaboratory and glasshouse studies were conducted to examine the leaf surface characteristics of selected weed species of oil palm. The broadleaf weeds selected were Asystasia gangetica, Borreria latifolia, Cleome rutidosperma, Clidemia hirta, Diodia ocimifolia and Mikania micrantha, while for the narrow leafs, Axonopus compressus, Cyperus kylingia, Eleusine indica, Paspalum conjugatum and Pennisetum polistachyon were investigated. The weeds were categorized into different types of roughness based on the macroscopic roughness, microscopic roughness and the estimation of three roughness parameters: Ra (arithmetic average height parameter), Rq (root-mean-square roughness parameter, corresponding to Ra), and Rz (average of high peaks and low valleys over the evaluation length). The leaf was examined using scanning electron microscopy (SEM) for the surface roughness, while the epicuticular wax content of the leaf was extracted using chloroform. The amount of wax extracted from the weeds varied between species. For broadleaf plants, Mikania micrantha (44.22 µg/cm2) was identified as the plant that contained the highest quantity of wax. Clidemia hirta (24.03 µg/cm2) and Asystasia gangetica (23.03 µg/cm2) were grouped in the plants with a medium quantity of wax while Cleome rutidosperma (16.52 µg/cm2), Borreria latifolia (14.19 µg/cm2) and Diodia ocimifolia (10.75 µg/cm2) were grouped in the plants with a low quantity of cuticular wax weight. For narrow leaf plants, Eleusine indica (44.23 µg/cm2) and Imperata cylindrica (49.88 µg/cm2) were recognized as the plants that contained a high quantity of wax. Pennisetum polystachion (32.16 µg/cm2) and Cyperus kylingia (22.85 µg/cm2) were categorized under the plants with a medium quantity of wax, whereas Paspalum conjugatum (19.59 µg/cm2) and Axonopus compressus (16.78 µg/cm2) were classified under the plant with a low quantity of wax. The wax on the abaxial surface data of the broadleaf weeds was found to be significantly different when compared to the adaxial surface data. In contrast, the amount of wax on the abaxial and adaxial leaf surface of the narrow leaf weeds was more or less similar. For the leaf surface roughness of the broadleaf species, Borreria latifolia was categorized as the roughest, followed by Clidemia hirta, Diodia ocimifolia, Asystasia gangetica and Cleome rutidosperma. Mikania micrantha had the smoothest leaf surface among the broadleaf species. On the other hand, the narrow leaf of Pennisetum polistachyon was identified as the roughest, followed by Imperata cylindrica and Paspalum conjugatum, while Eleusine indica, Axonopus compressus and Cyperus kylingia were categorized as having the smoothest leaf surface. Keywords : Leaf surface characteristic, roughness, wax amount Kajian makmal dan rumah kaca telah dilakukan untuk mengkaji kriteria permukaan daun rumpai terpilih dari ladang kelapa sawit. Rumpai daun lebar yang dipilih ialah Diodia ocimifolia, Borreria latifolia, Clidemia hirta, Cleome rutidosperma, Mikania micrantha dan Asystasia gangetica, sementara rumpai daun tirus yang dikaji ialah Eleusine indica, Cyperus kylingia, Axonopus compressus, Pennisetum polistachyon dan Paspalum conjugatum. Rumpai telah dikategori berdasarkan kepada kekasaran makroskopik, kekasaran mikroskopik dan parameter anggaran kekasaran: Ra (parameter purata ketinggian aritmetik), Rq (punca kuasa dua nilai parameter kekasaran, bersepadanan dengan Ra), dan Rz (purata tinggi dan rendah puncak lembah ke atas nilai panjang). Daun telah diperiksa dengan menggunakan mikroskopi imbasan elektron (SEM) untuk menilai kekasaran permukaan, sementara lilin epikutikular daun diekstrak dengan menggunakan kloroform. Kandungan lilin yang diekstrak daripada rumpai adalah bervariasi di antara spesis. Untuk tumbuhan daun lebar, Mikania micrantha (44.22 µg/cm2) telah dikenalpasti sebagai tumbuhan yang mengandungi jumlah lilin tertinggi. Clidemia hirta (24.03 µg/cm2) dan Asystasia gangetica (23.03 µg/cm2) dikategorikan sebagai tumbuhan dengan jumlah lilin yang sederhana, sementara Cleome rutidosperma (16.52 µg/cm2), Borreria latifolia (14.19 µg/cm2) dan Diodia ocimifolia (10.75 µg/cm2) dikategorikan sebagai tumbuhan dengan kuantiti lilin kutikular yang rendah. Untuk tumbuhan daun tirus, Eleusine indica (44.23 µg/cm2) dan Imperata cylindrica (49.88 µg/cm2) diakui mempunyai jumlah lilin terbanyak. Pennisetum polystachion (32.16 µg/cm2) dan Cyperus kylingia (22.85 µg/cm2) dikategorikan sebagai tumbuhan yang mempunyai jumlah lilin sederhana, manakala Paspalum conjugatum (19.59 µg/cm2) dan Axonopus compressus (16.78 µg/cm2) berada dalam kategori tumbuhan yang mempunyai jumlah lilin yang rendah. Jumlah lilin pada permukaan atas dan bawah daun bagi tumbuhan berdaun lebar mempunyai perbezaan yang nyata. Sebaliknya, jumlah lilin pada permukaan atas dan bawah bagi rumpai daun tirus adalah hampir bersamaan. Untuk nilai kekasaran permukaan daun daripada tumbuhan berdaun lebar, Borreria latifolia dikategorikan sebagai paling kasar diikuti oleh Clidemia hirta, Diodia ocimifolia, Asystasia gangetica dan Cleome rutidosperma. Mikania micrantha mempunyai permukaan daun yang paling halus di antara spesis daun lebar. Bagi tumbuhan berdaun tirus, Pennisetum polistachyon telah dikenalpasti sebagai paling kasar, diikuti oleh Imperata cylindrica dan Paspalum conjugatum, sementara Eleusine indica, Axonopus compressus dan Cyperus kylingia dikategorikan sebagai mempunyai permukaan daun yang halus. Kata kunci: Ciri permukaan daun, kekasaran, jumlah lili
Production of Phytate-Degrading Enzyme from Malaysian Soil Bacteria Using Rice Bran Containing Media
Get PDFThe aims of the study were to observe the effects of different concentration of rice bran in different media on phytase synthesis and to optimize the temperature and pH of the media for phytase production by those bacterial strains. Three bacterial strain isolates obtained from the soil of Malaysian maize plantation were used to produce phytase. In this study, the effects of different rice bran concentration, incubation temperature and initial pH-values of the media on phytase production were evaluated. Incorporation of 7.5% rice bran has the inducible effect on all the bacterial tested. In respect to phytase production, the best cultivation media and cultivation time for Bacillus cereus ASUIA260 was PFE with 7.5% rice bran after 3 days, whilst for Pantoea stewartii ASUIA271 and Enterobacter sakazakii ASUIA279, it was LB with 7.5% rice bran after 3 days and 5 days, respectively. The arrangement of those isolates according to their ability to produce phytases were E. sakazakii ASUIA279 > P. stewartii ASUIA271 > B. cereus ASUIA260. Production of phytase by those bacteria was triggered by the high content of organic phytate in the rice bran. Optimum temperature for phytase production of B. cereus ASUIA260 was 41 ºC compared to P. stewartii ASUIA271 and E. sakazakii ASUIA279 with 33 ºC and 37 ºC, respectively. Optimum initial pH for phytase production of B. cereus ASUIA 260 was pH 7.2, while P. stewartii ASUIA271 and E. sakazakii ASUIA 279 were both at pH 6.0
Molecular Phylogenetics of Cypripedium L. (Cypripedioideae: Orchidaceae) Based on Plastid and Nuclear DNA Sequences
Get PDFThe genus Cypripedium L. (Cypripedioideae: Orchidaceae) was in a state of taxonomic chaos for almost a century after the name was first established by Linnaeus. The beauty and scarcity of the plants has encouraged many taxonomists and botanists to investigate the relationships within Cypripedium. However, the studies often conflicted with each other and contributed towards difficulty in identification. In this study, a molecular phylogenetic analysis has been conducted on the two plastid DNA (rbcL and matK) and the nuclear DNA (ITS) in order to test the monophyly and intrageneric relationships in Cypripedium. The plastid data was combined with the nuclear ITS to further resolve the relationships among 38 investigated taxa of Cypripedium. Parsimony and Bayesian analyses indicated that the genus is monophyletic and C. irapeanum is sister to the rest of Cypripedium taxa. The phylogenetic results presented provide reliable information of intrageneric relationships in Cypripedium to be applied for future conservation studies. The present study confirmed the distinctiveness of all Endangered (C. farreri, C. fasciolatum, C. formosanum, C. franchetii, C. lichiangense and C. margaritaceum) and Critically Endangered (C. fargesii, C. henryi, C. smithii, C. wumegense and C. yunnanense) species listed in the International Union for Conservation of Nature, IUCN Red List of Threatened Species Database, thus supporting their worthiness for protection and conservation attention. Keywords: Conservation, Cypripedium, ITS, matK, molecular phylogenetic, rbcL Taksonomi genus Cypripedium L. (Cypripedioideae: Orchidaceae) telah berada dalam keadaan tidak stabil untuk hampir seabad lamanya selepas nama genus ini ditubuhkan oleh Linnaeus. Keindahan dan kepupusan tumbuhan ini telah mempergiatkan ramai pakar taksonomi dan ahli-ahli botani membuat penyiasatan terhadap hubungan-hubungan dalam Cypripedium. Walau bagaimana pun, kajian-kajian tersebut sering bercanggah di antara satu sama lain yang menyumbang ke arah kesukaran dalam pengenalpastian. Dalam kajian ini, suatu analisis filogenetik molekul telah dilaksanakan ke atas dua jenis plastid DNA (rbcL dan matK) dan satu DNA nukleus (ITS) bertujuan menguji monofili dan hubungan-hubungan intragenus dalam Cypripedium. Data plastid telah digabungkan dengan nuklear ITS untuk seterusnya menyelesaikan hubungan-hubungan antara 38 taksa Cypripedium. Analisis Parsimoni dan Bayesian menunjukkan Cypripedium adalah monofiletik dan C. irapeanum adalah kakak bagi keseluruhan taksa Cypripedium yang diuji. Hasil filogenetik menyediakan maklumat jelas hubungan-hubungan intragenus dalam Cypripedium untuk diaplikasikan dalam subjek pemuliharaan pada masa akan datang. Kajian mengesahkan sifat tersendiri semua spesis Terancam (C. farreri, C. fasciolatum, C. formosanum, C. franchetii, C. lichiangense dan C. margaritaceum) dan Sangat Terancam (C. fargesii, C. henryi, C. smithii, C. wumegense dan C. yunnanense) adalah tersenarai dalam ‘International Union for Conservation of Nature, IUCN Red List of Threatened Species Database’, seterusnya menyokong keperluan perlindungan dan perhatian terhadap pemuliharaan bagi spesis-spesis tersebut. Kata kunci: Pemuliharaan, Cypripedium, ITS, matK, filogenetik molekul, rbc
Surface Morphological Changes of Pathogenic Acanthamoeba spp. Treated with Mahanimbine
Get PDFAcanthamoeba spp. are the most common free-living amoebae (FLA) found abundantly in the environment. These opportunistic organisms are causative agents of Acanthamoeba keratitis, a painful eye infection that could result in loss of vision and is commonly associated with users of contact lenses. Two species of Acanthamoeba, namely Acanthamoeba sp. (SW isolate) and A. polyphaga (CCAP 1501/3A) were chosen in order to study the mechanism of action of a plant compound, mahanimbine, against these amoebae. Scanning electron microscopy (SEM) was used to analyze the cellular morphological changes that occurred to the amoebae after treatment with this pure compound at their IC50 concentration. The IC50 value for Acanthamoeba sp. (SW isolate) and A. polyphaga (CCAP 1501/3A) were 7.2 µg/mL and 1.00 µg/mL, respectively. The treatment was carried out for 72 hr at 30 °C and involved culturing the amoebae on cover slips in six-well culture plates, followed by normal processing methods of the amoebae for SEM. The evidences of damage on the trophozoites of amoebae were their rounded forms with cystic appearances and reduction in size. The number of acanthapodia decreased as compared to numerous spine-like acanthapodia as observed on untreated trophozoites. The research findings in the current study revealed that mahanimbine can cause cellular morphological changes especially to the cell shape and number of acanthapodia on the amoeba trophozoites. Keywords: Acanthamoeba, scanning electron microscopy (SEM), plant compounds, Acanthamoeba keratitis, mahanimbine Acanthamoeba spp. adalah ameba hidup bebas yang terdapat dengan banyak sekali di persekitaran. Organisma bersifat mengambil peluang ini adalah agen penyebab keratitis Acanthamoeba, sejenis jangkitan mata yang boleh menyebabkan kehilangan penglihatan dan biasanya berlaku kepada pengguna kanta lekap. Dua spesis Acanthamoeba, dinamakan sebagai Acanthamoeba sp. (isolat SW) serta A. polyphaga (CCAP 1501/3A), telah dipilih untuk kajian mekanisma tindakan satu sebatian tumbuhan iaitu mahanimbin terhadap kedua-dua spesis ameba ini. Mikroskop imbasan elektron (SEM) telah digunakan untuk menganalisis perubahan morfologi sel yang terdapat pada ameba setelah dirawat dengan mahanimbin pada kepekatan IC50 untuk setiap spesis. Nilai IC50 mahanimbin yang diperolehi untuk Acanthamoeba sp. (isolat SW) dan A. polyphaga (CCAP 1501/3A) ialah 7.2 µg/mL dan 1.00 µg/mL, secara berurutan. Rawatan telah dijalankan selama 72 jam pada suhu 30 °C yang melibatkan pengkulturan ameba di atas sisip kaca di dalam piring kultur enam lubang, diikuti dengan kaedah pemprosesan biasa ke atas ameba untuk SEM. Bukti-bukti kerosakan pada sel yang dapat dilihat ialah trofozoit-trofozoit ameba berbentuk bulat dan seperti sista. Saiz trofozoit-trofozoit ini juga berkurangan serta mengecut. Bilangan akantapodia juga berkurangan berbanding bilangan akantapodia seperti duri pada trofozoit yang tidak dirawat. Hasil kajian ini menunjukkan mahanimbin boleh menyebabkan perubahan morfologi terutamanya pada bentuk sel dan bilangan akantapodia pada trofozoit-trofozoit ameba. Kata kunci: Acanthamoeba, mikroskopi imbasan elektron (SEM), sebatian tumbuhan, keratitis Acanthamoeba, mahanimbi