METODE MULTIPLEX RT-PCR UNTUK DETEKSI SIMULTAN CRINIVIRUS DAN BEGOMOVIRUS PADA TANAMAN TOMAT

ESTI PRASETYA N, Dr. Ir. Sedyo Hartono, M.P; Dr. Ir. Sri Sulandari, S.U

2018 | Tesis | MAGISTER FITOPATOLOGI

Akhir-akhir ini banyak terjadi gejala menguning pada tanaman tomat (Lycopersicon esculentum Miller) dengan indikasi terancam infeksi campuran Crinivirus dan Begomovirus. Serangan kedua virus tersebut menginduksi gejala menguning pada daun dan menyebabkan buah menjadi kurang layak dipasarkan. Deteksi beberapa virus tanaman selama ini dilakukan secara terpisah. Salah satu metode deteksi cepat, akurat dan efisien beberapa virus tanaman secara simultan yaitu metode multiplex RT-PCR. Penelitian ini bertujuan untuk mendeteksi virus-virus yang berasosiasi dengan penyakit kuning pada tanaman tomat dan serangga vektornya serta mengoptimasi metode multiplex RT-PCR untuk deteksi simultan Crinivirus dan Begomovirus. Metode yang digunakan adalah survei lapangan, ekstraksi RNA/DNA dari tanaman dan serangga dengan beberapa metode, kuantifikasi RNA/DNA, sintesis cDNA, single RT-PCR dan PCR, multiplex RT-PCR, visualisasi hasil amplifikasi RT-PCR dan PCR serta analisa sekuen DNA. Hasil pengamatan lapangan di daerah Kopeng dan Ketep (Jawa Tengah), Pakem (Yogyakarta), Bogor (Jawa Barat) dan Malang (Jawa Timur) ditemukan adanya variasi gejala klorosis menyerupai gejala infeksi campuran Crinivirus dan Begomovirus dengan kejadian penyakit di atas 50% dan ditemukan populasi kutu kebul di sekitar pertanaman tomat. Berdasarkan pengujian laboratorium, metode ekstraksi dsRNA sederhana menghasilkan RNA virus murni sedangkan metode SDT dan kit komersial menghasilkan RNA total. Konsentrasi RNA dengan metode dsRNA sederhana dan SDT lebih rendah dibandingkan kit komersial, namun kedua metode dapat digunakan dalam deteksi rutin untuk preparasi RNA karena lebih efisien dibandingkan kit komersial. Ekstraksi DNA tanaman menggunakan kit komersial menghasilkan DNA total dimana konsentrasi DNA tanaman yang diperoleh cukup tinggi dan memiliki kualitas sangat baik. Hasil multiplex RT-PCR menggunakan tiga pasang primer spesifik dengan suhu annealing 58 oC berhasil mengamplifikasi tiga virus secara simultan pada sampel tanaman dari Kopeng, Ketep dan Bogor. Hal ini menunjukan bahwa tehnik multiplex RT-PCR sesuai untuk deteksi simultan TICV, ToCV dan Begomovirus pada tanaman tomat. Hasil sekuen DNA dari sampel tanaman diperoleh TICV dan ToCV untuk Crinivirus serta PepYLCIV dan ToLCNDV untuk Begomovirus. Hasil single RT-PCR maupun multiplex RT-PCR serangga T. vaporariorum menunjukan pertanaman tomat di daerah Ketep dan Kopeng dapat terinfeksi oleh TICV dan/atau ToCV. Sedangkan untuk hasil single PCR maupun multiplex RT-PCR serangga B. tabaci menunjukan pertanaman tomat di daerah Pakem dapat terinfeksi oleh Begomovirus dan/atau ToCV.

Lately, there have been a lot of yellowing symptoms on tomato plants (Lycopersicon esculentum Miller) with indications of being threatened by a mixed infection with Crinivirus and Begomovirus. The attacks of both viruses induce yellowing of the leaves and cause the fruit to be less marketable. Detection of several plant viruses has been carried out separately. One method of rapid, accurate and efficient detection of several simultaneous plant viruses is the multiplex RT-PCR method. This study aims to detect viruses associated with yellowing diseases in tomato and insect vector plants and optimize the multiplex RT-PCR method for simultaneous detection of Crinivirus and Begomovirus. The methods used were field survey, extraction of RNA / DNA from plants and insects with several methods, RNA / DNA quantification, cDNA synthesis, single RT-PCR and PCR, multiplex RT-PCR, visualization of RT-PCR and PCR amplification results and sequence analysis. DNA. Field observations in the areas of Kopeng and Ketep (Central Java), Pakem (Yogyakarta), Bogor (West Java) and Malang (East Java) found variations in symptoms of chlorosis resembling the symptoms of mixed infections with Crinivirus and Begomovirus with disease incidence above 50% and found whitefly populations around tomato plants. Based on laboratory testing, simple dsRNA extraction methods produce pure viral RNA while the SDT method and commercial kits produce total RNA. RNA concentrations with simple dsRNA methods and SDT are lower than commercial kits, but both methods can be used in routine detection for RNA preparation because it is more efficient than commercial kits. DNA extraction of plants using a commercial kit produces total DNA in which the DNA concentration of plants obtained is quite high and has very good quality. The results of multiplex RT-PCR using three pairs of specific primers with annealing temperature of 58 oC succeeded in amplifying three viruses simultaneously on plant samples from Kopeng, Ketep and Bogor. This shows that the multiplex RT-PCR technique is suitable for simultaneous detection of TICV, ToCV and Begomovirus in tomato plants. The DNA sequences from plant samples were obtained by TICV and ToCV for Crinivirus and PepYLCIV and ToLCNDV for Begomovirus. Single RTPCR results and T. vaporariorum insect multiplex RT-PCR showed that tomato planting in Ketep and Kopeng areas could be infected by TICV and/or ToCV. Whereas for single PCR and multiplex RT-PCR results, B. tabaci showed that tomato planting in Pakem area could be infected by Begomovirus and/or ToCV.

Kata Kunci : Begomovirus, Crinivirus, dsRNA, multiplex RT-PCR, SDT