Loop mediated isothermal amplification (LAMP) assay for detection of coconut root wilt disease and arecanut yellow leaf disease phytoplasma.
The coconut root wilt disease (RWD) and the arecanut yellow leaf disease (YLD) are two major phytoplasma associated diseases affecting palms in South India. Greatly debilitating the palm health, these diseases cause substantial yield reduction and economic loss to farmers. A rapid and robust diagnostic technique is crucial in efficient disease management. We established phytoplasma 16S rDNA targeted loop mediated isothermal amplification (LAMP) and real time LAMP based diagnostics for coconut RWD and arecanut YLD. The LAMP reaction was set at 65 °C and end point detection made using hydroxynaphthol blue (HNB) and agarose gel electrophoresis. Molecular typing of LAMP products were made with restriction enzyme HpyCH4 V. Conventional PCR with LAMP external primers and sequencing of amplicons was carried out. Real time LAMP was performed on the Genei II platform (Optigene Ltd., UK). An annealing curve analysis was programmed at the end of the incubation to check the fidelity of the amplicons. The phytoplasma positive samples produced typical ladder like bands on agarose gel, showed colour change from violet to blue with HNB and produced unique annealing peak at 85 ± 0.5 °C in the real time detection. Restriction digestion produced predicted size fragments. Sequencing and BLASTN analysis confirmed that the amplification corresponded to phytoplasma 16S rRNA gene. LAMP method devised here was found to be more robust compared to conventional nested PCR and hence has potential applications in detection of phytoplasma from symptomatic palm samples and in rapid screening of healthy seedlings.
Central Plantation Crops Research Institute, Indian Council of Agricultural Research (ICAR), Kudlu P.O, Kasaragod, 671124, Kerala, India.
Smita
S
Nair
Department of Biotechnology, Sugarcane Breeding Institute, Indian Council of Agricultural Research (ICAR), Coimbatore, 641007, Tamil Nadu, India. rmanimekalaiicar@gmail.com.
Ramaswamy
R
Manimekalai
Central Plantation Crops Research Institute, Indian Council of Agricultural Research (ICAR), Kudlu P.O, Kasaragod, 671124, Kerala, India.
Palliyath
P
Ganga Raj
Central Plantation Crops Research Institute, Indian Council of Agricultural Research (ICAR), Kudlu P.O, Kasaragod, 671124, Kerala, India.
Vinayaka
V
Hegde
D001114:Areca / Q000382:microbiology*; D001431:Bacteriological Techniques / Q000379:methods; D003059:Cocos / Q000382:microbiology*; D004269:DNA, Bacterial / Q000235:genetics; D004587:Electrophoresis, Agar Gel; D007194:India; D021141:Nucleic Acid Amplification Techniques / Q000379:methods; D045582:Phytoplasma / Q000235:genetics / Q000302:isolation & purification*; D010935:Plant Diseases / Q000382:microbiology*; D018515:Plant Leaves / Q000382:microbiology; D018517:Plant Roots / Q000382:microbiology; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D017422:Sequence Analysis, DNA
D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S
16S rRNA gene; LAMP; Phytoplasma; Real time LAMP