Accurate and sensitive detection of areca palm yellow leaf phytoplasma in China by droplet digital PCR targeting tuf gene sequence

Abstract

Droplet digital PCR (ddPCR) is a method for qualitative and quantitative detections of nucleic acid molecules with high sensitivity. In this study, a ddPCR detection system for areca palm yellow leaf (AYL) phytoplasma was developed using the primers Atf/Atr and probe AtProbe, which were designed based on the tuf gene of the phytoplasma. AYL phytoplasma-infected samples were collected from different regions in Hainan Province of China and the concentration distribution of AYL phytoplasma in the samples was detected and analysed by the ddPCR detection system. The results indicated that the phytoplasma was significantly detected in the AYL sample with the primers Atf/Atr and the probe AtProbe in ddPCR, and the number of droplets generated in each ddPCR reaction system was more than 10,000, indicating that the absolute quantitative results were reliable. Using the ddPCR assay for AYL phytoplasma, the phytoplasma was detected in the AYL samples collected from different regions such as Ding'an, Sanya, Tunchang, Wenchang and Wanning counties in Hainan Province of China. The lowest concentration of AYL phytoplasma that was detected by ddPCR method was 0.07 copies Á μl À1 . Compared with LAMP detection, the sensitivity of ddPCR detection was improved about 1,000 times better. The concentration distribution of the AYL phytoplasma in the same leaf from the same diseased areca palm was significantly uneven (p < 0.05). The ddPCR detection system established based on the tuf gene of phytoplasma in the study would be valuable for the qualitative and quantitative detections of AYL phytoplasma, and it will also assist in the study of the epidemiology and management of the disease.areca palm yellow leaf disease, droplet digital PCR, molecular detection, phytoplasma, tuf gene | INTRODUCTIONPhytoplasmas are prokaryotic pathogens that parasitize the phloem of plants and cannot be cultured in vitro (Doi, Teranaka, Yora, & Asuyama, 1967). Areca palm (Areca catechu L.) is an evergreen tree belonging to the palm family, with high medicinal and economic value.Areca palm yellow leaf (AYL) disease associated with a phytoplasma is a devastating disease of areca palm. AYL diseases have been reported