Tri Puji Priyatno

Keragaman Genetik Rizobakteri Penghasil Asam Indol Asetat Berdasarkan 16S rRNA dan Amplified Ribosomal DNA Restriction Analysis

Asam indol asetat (AIA) dapat dihasilkan oleh bakteri rizosfer/rizobakteri pemacu pertumbuhan tanaman (PPT). Keragaman genetik isolat bakteri PPT indigenous Indonesia perlu diinvestigasi untuk mencari sumber potensial agen PPT dengan informasi kekerabatan intra dan interspesies yang jelas. Karena itu penelitian ini bertujuan mengetahui keragaman genetik rizobakteri penghasil AIA indigenous Indonesia dengan gen 16S rRNA, dilengkapi dengan ARDRA. Koleksi isolat bakteri BB Biogen diidentifikasi kandungan AIA-nya, morfologi secara makroskopis dan sekuensing pada sekuen 16S rRNA dan ARDRA. Total empat belas isolat rizobakteri memiliki kandungan AIA dalam kisaran 5,24-37,69 µg/ml dan tertinggi pada SM1. Karakteristik morfologi koloni rizobakteri mendukung variasi strain bakteri penghasil AIA. Delapan isolat terpilih diidentifikasi sebagai spesies Bacillus dengan homologi 96-99%. Lima isolat (SM1, JP4, KP3, MB2, dan CP3) diidentifikasikan sebagai B. subtilis , SC2 sebagai B. amyloliquefaciens , BL2 dekat dengan B. velezensis, dan JP3 memiliki homologi tinggi dengan Brevundimonas olei. Delapan isolat rizobakteri tersebut berkerabat dekat dengan strain bakteri referensi yang memiliki kesamaan spesies. Analisis ARDRA- Rsa I menghasilkan lima filotipe dengan keunikan pola sidik jari. Isolat CP3, MB 2, dan KP 3 berada dalam satu filotipe. Kedekatan isolat dalam Bacillus sp. digambarkan oleh filotipe 5 ( B. subtilis SM1 dan B. velezensis BL2) yang diduga jauh dari B. amyloliquefaciens SC2 (filotipe 4) dan JP 3 pada genus Brevundimonas (filotipe 3). Keragaman genetik isolat rizobakteri penghasil AIA terhitung rendah berdasarkan 16S-rRNA dan ARDRA- Rsa I.

Control of Anthracnose Disease (Colletotrichum gloeosporioides) Using Nano Chitosan Hydrolyzed by Chitinase Derived from Burkholderia cepacia Isolate E76

Anthracnose (Colletotrichum gloeosporioides) is one of the important diseases of fruit crops that need to be controlled. This study was aimed to obtain the best formula of hydrolyzed nano chitosan and its potensial in controlling anthracnose. The hydrolyzed chitosan was prepared using chitinase enzyme extracted from Burkholderia cepacia isolate E76. Chitosan nanoparticles were synthesized using ionic gelation method by reacting hydrolyzed chitosan (0.2%) with Sodium tripolyphosphate (STPP) (0.1%) as cross-linking agent using 30n60 minutes stirring condition. The bioactivity of the nano chitosan formula was tested to C. gloeosporioides under in vitro and in vivo assays. The specific enzymatic activity of the purified chitinase was higher (0.19 U/mg) than that of crude enzyme (supernatant) with the purity increased by 3.8 times. Of the four formula tested, Formula A (hydrolyzed chitosan to STPP volume ratio of 5 : 1 with 60 minutes stirring condition) was found good in terms of physical characteristic of the particle. The formula nano chitosan particle had the spherical-like shape with an average particle size of 126.2+3.8 nm, polydispersity index (PI) of 0.4+0.02, and zeta potential (ZP) value of 27.8+0.2 mV. Nano chitosan had an inhibitory activity to C. gloeosporioides in vitro up to 85.7%. Moreover, it could inhibit 61.2% of C. gloeosporioides spores germination. It was shown that nano chitosan was also effective to reduce anthracnose disease severity in vivo when applied as a preventive measure on chili and papaya fruits. This study could be used as a reference for further fruit coating application using nano chitosan as a promising postharvest biocontrol agent to C. gloeosporioides.

Resistance and Phenotypic Character of Chili M2 Mutant Lines Against Chilli Veinal Mottle Virus

Chilli veinal mottle virus infection (ChiVMV) could reduce the quality and 60–100% of yield losses of chili. Among the chili varieties released, no one has been resistant to ChiVMV, mainly due to a high variation of ChiVMV strains and not well mapped. Therefore, finding a new source of ChiVMV resistant genes is pivotal role in order to assembly new varieties. Approach through in vitro mutation induction using mutagen ethyl methane sulfonate (EMS) is one of the efforts to increase genetic diversity. Previous studies has successfully acquired 800 M2 lines through callus induction of Gelora variety with EMS. This study aimed to obtain M2 lines resistant to ChiVMV and having a good agronomical characters. A total of 800 chili M2 lines that derived from chili M2 mutations using mutagen EMS has been tested in greenhouse to ChiVMV resistance and studied character phenotype. The results showed that of the 800 lines, there were 28 strains obtained showed a response tolerant and resistant to ChiVMV. Eight mutant lines of which have good agronomic characters. The mutant lines are M2.100, M2.108, M2.200, M2. 122, M2.238, M2.353, M2.420, and M2.517. Eight lines will be selected and further observed to obtain chili promising lines that are resistant to ChiVMV and high yielding.

Karakterisasi dan Identifikasi Isolat Bakteri Endofitik Penghambat Jamur Patogen Padi

Local variety is the major component in upland rice cultivation as well as breeding material in variety improve-ment. Local upland rice varieties have been adapted to specific location, preferred and adopted by farmers although they are late in maturity. These reasons make it majority of farmers in West Pasaman District (West Sumatra) still cultivate local upland rice varieties. To know the yield potential and agronomic characters of widely cultivated of upland rice in West Pasaman District, a variety trial conducted in 2010. Five local varieties and one improve variety planted a Randomized complete block arrangement with three replications. Observa-tion conducted on agronomic characters and yield and yield components. The results showed that local variteies were tall (153-171 cm) and late maturity (130-157 days). The local varieties also showed higher number of spikelet/panicle (249-454) lighter 1.000 grains weight (<20 g) and higher yield than improved variety (4.27-5.51 compared to 2.85 t/ha). In general all plant characters showed high heritability estimate (except number of productive tiller/hillr). High estimated of genetic variation coefficient and genetic advance were shown on number of grain per panicle. Based on these genetic parameters, selection at early generation for high yield can conducted on number of grain per panicle.Phosporus (P) deficiency is one of limiting factor for rice growth. In Indonesia P deficiency much occurs in acid soils. The use of P deficiency tolerant varieties is the best solution compared to the application of P fertilizer due to more efficient in cost. The purpose of this study was to evaluate the local rice germplasm collected from several regions in Indonesia to P deficiency in acid soil. The study was conducted in Jasinga West Java during wet season of 2006- 2007 with the soil condition lack of P. The experiments were conducted with the two treatments: first, without P fertilizer and second with P fertilizer equivalent of 25 kg P/ha. The total 100 accessions of rice germplasm were screened in this field based on a randomized block design with three replications. Fertilizing were given on experiments I and II with the composition: urea 300 kg/ha and 100 kg KCl. The field design was done by plotting size is 1 x 5 m2, spacing plant of 25 cm x 25 cm, and planting two seeds per hole. The yield components characters which were observed: number of tillers, plant dry weight, plant height and flowering. The results of combined analysis showed that there is a significant interaction between P and genotype on the tiller number, whereas the dry weight of plant, plant height, and flowering were is not significant. The evaluation of 100 local rice genotypes to P deficiency by indicators of the tiller number and dry weight of plants obtained 19 genotypes that are tolerant to P deficiency with the relative value of tillers number and dry weight of plants more than 80%. Local varieties Mandalet, Ganefo, Padi Belanda, Pulut Jangan, Padi Ubek Bala, and Padi Krayan were the most tolerant to conditions without P which showed the tillers number more 1-21% rather than the conditions with P fertilizer. There are three selected genotypes, Pulut Jangan, Padi Ubek Bala, and Padi Krayan which increased the number of tillers and dry weight of plants in without P conditions. The increasing of tillers number were reach 1-17% while the dry weight increased 12-41%.

Identifikasi Gen elF4G asal Oryza rufipogon pada Padi Varietas Inpari HDB dan Inpari Blas

Wild rice accession Oryza rufipogon is known as a source of tungro resistance genes and has been used to develop tungro resistant varieties, especially against rice tungro spherical virus (RTSV). However the genes have not been identified yet. Previously, an eukaryotic Translation Initiation Factor (eIF4G) was identified as a putative gene associated with recessive resistance gene against RTSV in Utri Merah variety. The research was aimed to detect and identify the eIF4G gene on rice var.Inpari HDB and var. Inpari Blas using specific primer that was designed based on Utri Merah eIF4G gene sequences. The resistance respons of both Inpari varieties and O.rufipogon against 3 tungro virus isolates were conducted in the green house trial. Assays were done based on international rice testing nursery according to IRRI. The eIF4G gene is amplified by PCR and the amplicon was directly sequenced, then analysed in silico. The results showed that all 3 varieties are classified as resistant against tungro virus isolates. PCR was successfully amplified the eIF4G gene with size ~300 bp in both of varieties and their parent O. rufipogon . The nucleotides homology of eIF4G among those 2 varieties and O. rufipogon is up to 100%, while the homology to Utri Merah was 93%. There were 4 nucleotides deletion and 16 nucleotides differences between Utri Merah and those 2 varieties and O. rufipogon , respectively. Those nucleotide differences lead to deletion of 1 amino acid and 4 amino acids different between both Inpari varieties and O.rufipogon in comparison with corresponding amino acid in Utri Merah.

KARAKTERISASI ß-1,3-1,4-GLUKANASEBAKTERI ENDOFITIK Burkholderia cepacia ISOLATE76 ASAL TANAMAN PADI

Pathogenic fungus is one of the constraints to increase crop production. Chemical control using fungicides caused negative effects either to the environment or increased pathogen resistance to fungicide. Biological control using microbial-producing s glucanase is an alternative method to inhibit the growth of pathogenic fungus. The aim of this study was to characterize s-1,3-1,4-glucanase produced by rice endophytic bacterium, B. cepacia E76. Purification was carried out by ammonium sulphate precipitation, dialysis, and ion exchange chromatography using DEAE sepharose Fast Flow. A further characteristic of the enzyme activity was studied using oatmeal-glucan substrate.Results showed that precipitation using saturated 80% ammonium sulphate generated a good yield with the purity increased by 11 fold and yield of 66%.After chromatography step, the s-1,3-1,4-glucanase of B. cepacia was successfully purified with an increasedof purity up to 33 fold and yield of 4%. Based on 10% SDS-PAGE, the enzyme profiles had the molecular weight of 15, 48 and 55 kDa.Of the three isozymes, only the 48 kDa isozyme showed the strongest glucanase activity when grown on media containing glucan as substrate.