Abstract. Thesis (2010), Master s program In Biology, School of Live Sciences and Technology-ITB,

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2 Virulence selection of Phytophthora infestans ( Mont. ) de Bary population in West Java to transformed potato plant (Solanum tuberosum L.) containing RB gene Student : Hani Susanti Thesis (2010), Master s program In Biology, School of Live Sciences and Technology-ITB, h_ni2003@yahoo.com Advisors : Dr. Rizkita Rachmi Esyanti 1 and Dr. Eri Sofiari 2 1 School of Live Sciences and Technology-ITB, rizkita@sith.itb.ac.id 2 Indonesian Vegetable Research Institute, esofiari@indosat.net.id Degree : Magister Sains (M.Si), Conferred July 2010 Abstract Phytophthora infestans (Mont.) de Bary causes late blight (LB) disease that can decrease potato production up to 100 %. These oomycete has two mating types, namely A1 and A2. Oospores, as a generative structure of P. infestans, are formed by mating between A1 and A2. Sexual reproduction generates more genetic variation that will be more difficult to control. Resistance cultivars in potato (Solanum tuberosum L.) to LB is a potential important factor in controlling this disease. RB gene as source of resistance could be obtained by genetically modifying potato cultivars with resistance genes from wild species Solanum bulbocastanum. These genes were inserted by using transformation techniques and subsequent hybridization between Granola or Atlantic with transformed plant (hybrid plant). The research was conducted to identify mating type of P. infestans isolates which were collected from West Java, followed by testing degree of virulence of the isolates to the transformed and hybrid potato plants containing Rblb genes. Phytophthora infestans was isolated from leaves infected by LB from 5 area in Bandung, 4 area in Garut and one area in Cianjur. Isolates were cultured on V8 - agar medium at 18 ± 2 0 C to enable the test of mating type. Test of virulence and selection to virulence were performed using a detached leaves assay on water - agar medium. Plants were grouped into three categories, namely the transformed potato plants containing RB gene, hybrid plants, and the Atlantic & Granola potato varieties as a control. The degree of plant resistance was calculated based on disease severity tested in the experimental station. Microscopic analysis of mating-type test showed that only very few oospore structures were found in several isolates, so that they were suspected as potentially self-fertile, although there was a possibility to be grouped in the A1 & A2 mating type. Based on statistic analysis, the average leaf infected by P. infestans isolated from Bandung, Cianjur and Garut in potatoes containing the RB gene was significantly lower than potatoes in control group. Results showed that 48% of potato leaves containing RB gene suffered from LB, whereas the control group was infected 85-99%. The level of

3 virulence of P. infestans isolates on potato plants containing RB gene is still low compare to that in the control group. This shows that the RB gene is a potential source of resistance on potato plants against LB disease. Keywords : Phytophthora infestans, Solanum tuberosum, late blight, RB genes, transformation, cisgenic, virulence level, mating type

4 Seleksi virulensi populasi Phytophthora infestans ( Mont. ) de Bary di Jawa Barat terhadap tanaman kentang (Solanum tuberosum L.) hasil tarnsformasi dengan gen RB Mahasiswa : Hani Susanti Tesis (2010), Program Studi Magister Biologi SITH, h_ni2003@yahoo.com Pembimbing : Dr. Rizkita Rachmi Esyanti 1 dan Dr. Eri Sofiari 2 1 SITH-ITB, rizkita@sith.itb.ac.id 2 Balai Penelitian Tanaman Sayuran, esofiari@indosat.net.id Gelar : Magister Sains (M.Si), Wisuda Juli 2010 Abstrak Phytophthora infestans (Mont.) de Bary merupakan jamur oomycetes penyebab penyakit hawar daun pada kentang yang dapat menurunkan produksi hingga mencapai 100%. Jamur ini dikelompokkan dalam 2 tipe perkawinan (mating type) yaitu A1 dan A2. Struktur generatif oospora akan terbentuk melalui perkawinan A1 dan A2, sehingga banyak terjadi keragaman dan semakin sulit dikendalikan. Salah satu upaya pengendalian penyakit hawar daun adalah dengan menggunakan kentang resisten. Sifat resistensi kentang dapat diperoleh dengan menyisipkan gen RB dari kerabat kentang liar Solanum bulbocastanum melalui metoda transformasi, dan dilanjutkan dengan hibridisasi antara kentang yang mengandung gen RB dengan kentang kultivar Granola atau Atlantik. Penelitian ini bertujuan untuk melakukan identifikasi mating type pada isolat P. infestans dari beberapa tempat di Jawa Barat dan seleksi virulensi isolat tersebut terhadap tanaman kentang transgenik yang mengandung gen RB. Isolat P. infestans diperoleh dari sampel daun yang terinfeksi pada 5 lokasi di Bandung, 4 lokasi di Garut dan 1 lokasi di Cianjur. Isolat dikultur pada suhu 18±2 0 C dan dilakukan pengujian mating type pada media V8 agar. Uji virulensi dan seleksi virulensi dilakukan menggunakan metoda Detached Leaves Assay pada medium air agar. Tanaman uji dikelompokkan menjadi 3, yaitu tanaman kentang hasil transformasi gen RB, hibrid, serta kentang kultivar Atlantik dan Granola sebagai kontrol. Tingkat resistensi tanaman dihitung berdasarkan pada data intensitas serangan P. infestans di lapangan uji terbatas (LUT). Hasil pengamatan mikroskopis menunjukkan bahwa hanya ditemukan sedikit sekali struktur generatif oospora pada uji mating type sehingga isolat diduga melakukan fertilisasi sendiri (selffertilization) atau dapat dikelompokkan pula pada tipe perkawinan A1&A2, dengan rata-rata tingkat virulensi tidak berbeda nyata antara isolat Bandung, Cianjur dan Garut. Berdasarkan uji statistik ANOVA dengan selang kepercayaan 95% diperoleh bahwa rata-rata daun yang terinfeksi oleh isolat Bandung, Cianjur dan Garut pada kentang yang mengandung gen RB berbeda nyata dengan kentang kelompok kontrol. Hasil seleksi virulensi menunjukkan 48 % daun kentang yang i

5 mengandung gen RB terserang hawar daun, sedangkan daun kentang kelompok kontrol terserang 85-99%. Tingkat virulensi isolat-isolat tersebut terhadap kelompok kentang yang mengandung gen RB masih rendah dibandingkan kelompok kontrol. Hal ini menunjukkan bahwa gen RB dapat digunakan sebagai sumber ketahanan tanaman kentang terhadap penyakit hawar daun. Kata kunci : Phytophthora infestans, Solanum tuberosum, hawar daun, gen RB, transformasi, cisgenik, tingkat virulensi, mating type ii