Investigation Of Rice Orange Leaf Disease In Southern China And Establishment Of Its Molecular Detection Method

Rice orange leaf disease(ROLD) caused by a phytoplasma was historically outbreak in parts of southern China during the late 1980 s to the early 1990 s. After that, it was declined in the past more than 10 years. A recent research by our team discovered that this disease can be transmitted by the leafhopper nephotettix cinticeps efficiently, which is a novel vector in addition to the unique vector leafhopper inazuma dorsalis reported previously. It is necessary to monitor the epidemic dynamics and establish the rapid detection technology of the disease because of the ability of new vector. Therefore, this study carried out extensive field investigation in South China, made clear the disease distribution and occurrence status; obtained the bacteria near full-length genome sequences by Next Generation Sequencing method; developed the specific PCR detection technology based on cell division protein gene; and established the synchronous detection of rice orange leaf phytoplasmas(ROLP) and rice gall dwarf virus(RGDV) in rice plant and single vector leafhoppers by dual one-step RT-PCR detection method. The main results as follows:1?The distribution and occurrence status of ROLD in Southern China were defined.Field investigation in South China at 2015 and 2016 early rice period showed that the occurrence of ROLD were significantly expanded, except the southwest Guangdong and the southeast Guangxi which were the ROLD perennial occurrence areas, we also discovered the disease in western Guangdong, northern Guangdong and the Pearl River Delta region and the central Hainan, parts of the regions occured seriously, Luoding city in southwest Guangdong suffered the most. Combine the investigation of field disease transmission vector, especially new vector nephotettix cincticeps, predicting the ROLD will be very likely a epidemic disaster in the South China rice area.2?Obtained the near full length of Rice orange leaf phytoplasma genome nucleotide sequence. Carried out semi quantitative Nest PCR, primers based on pathogen 16 S r DNAsequence were designed, the results showed that, the rice leaf sheath tissue with markedly symptomatic at tillering stage possess the highest pathogen content, was the main vein of the infected leaves secondly, was leaf tissues again. Took rice plants sheath tissue with apparent ROLD symptoms to extract total DNA, then interrupted into fragments randomly by ultrasonic treatment, deep sequencing of the total DNA was carried out using Illumina Hiseq TM2000 technology, received 6.525 G clean reads. We gained 60 contigs(average length 9389nt) after selecting and assembling, and finally got about 607385 bp frame sequence of ROLD genome using Candidatus Phytoplasma asteris line onion yellows- mild whole genome as a reference, with the utilizing of bioinformatics software, suggesting that the frame sequence account for more than 70% of it's full length genome.3?An ordinary PCR detection method of rice orange leaf disease and a dual one-step RT-PCR detection method for simultaneous detection of ROLP and RGDV was established.Chose ROLP infection required cell division protien gene as a target, specific primers were designed, then developed the PCR detection method of ROLD, the size of amplified products was 492 bp which can be clearly distinguished by agarose gel electrophoresis, the sensitivity were 10-3 per 100 mg of infected rice plant tissue DNA extracts and 10-1 per single leafhopper DNA extracts. ROLD and RGDV were transmitted by inazuma dorsalis and nephotettix cincticeps, the two diseases often occurred in mixed, this research used cell division protien gene of ROLP and P8 gene of RGDV established a dual one-step RT-PCR method to detection the two pathogen in rice plant and leafhopper samples. The method obtained corresponding pathogen gene fragments from rice plant tissues and leafhopper total RNA extracts specifically.