Identification and multiplex reverse transcription polymerase chain reaction detection of viruses associated with mosaic symptom on patchouli (Pogostemon cablin Benth.).

Abstract

Patchouli essential oil is one of sources of Indonesia foreign exchange, since Indonesia is the biggest supplier of patchouli oil in the world. One of obstacle on the patchouli production is viral infection. Virus(es) infecting patchouli plant has not been well known. Thus, the aim of presented work is to detect and identify virus(es) associated with mosaic symptom on patchouli either serologically or molecularly. Samples were collected from patchouli cultivation in Cicurug and Cijeruk (Sukabumi) and in Manoko (Lembang, Bandung) by taking 30 symptomatic and asymptomatic samples from each location. Serological test using four antisera to Cucumber mosaic virus (CMV), Cymbidium mosaic virus (CymMV), Potyvirus and Broad bean wilt virus 2 (BBWV2) were carried out to determine the incidence of virus infection. Further molecular detection of virus(es) was carried out by reverse transcription polymerase chain reaction (RT-PCR) and DNA sequencing. Multiplex RT-PCR was developed to provide rapid detection of those viruses used eight combination of primer concentrations (I, II, III, IV, V, VI, VII and VIII). Serology detections showed that BBWV2 and Potyvirus were detected in all sampling locations, CymMV was found in Cicurug and Manoko, while CMV was detected in Cicurug and Cijeruk. Potyvirus disease incidence was 100% in Manoko, 83.3% in Cicurug and 50.0% in Cijeruk. BBWV2 disease incidence was 100% in Manoko, 73.3% in Cicurug and 90.0% in Cijeruk. Disease incidence of CymMV in Manoko and Cicurug was 3.3% , while the incidence of CMV in Cicurug was 80.0% and 13.3% in Cijeruk. Each of primer pairs were able to amplify the coresponding viruses, which confirmed the serology detections. Nucleotide and amino acid sequence homology analysis showed that BBWV2 isolated from Manoko has a highest homology with BBWV2 from Singapore (natural host: Megakepasma erythroclamys / ME). BBWV2 from Manoko clustered with BBWV2 from Singapore (ME), China (pepper) and South Korea (red pepper) based on the nucleotide and amino acid sequences. CymMV from Manoko has a highest homology of nucleotide and amino acid sequences with CymMV from South Korea (Cymbidium). CymMV from Manoko clustered with CymMV from India (Phaius sp), Indonesia (Dendrobium), China (Cymbidium and vanilla), Thailand (Oncidium), Hawai (Dendrobium) and South Korea (Cymbidium) based on nucleotide sequence. While based on amino acid sequence, CymMV from Manoko clustered with CymMV from India (Phaius sp and vanilla), and Singapore (Dendrobium). Multiplex RT-PCR was succesfully amplified Potyvirus, BBWV2 and CymMV by using primer combination V, VI, VII and VIII. The multiplex RT-PCR can be applied to detect multiple viruses infection on patchouli simultaneously.