Compared with traditional nested PCR, results suggested that the established ROLP detection method is more sensitive and time saving.įig. One set of specific primers was selected to establish an accurate PCR detection method for ROLP in rice and leafhopper vector samples. In this study, we designed six sets of primers based on the sequences of these three genes for testing in PCR. 2011 Mahillon and Chandler 1998 Ostermann et al. Genome sequence analysis showed the sequences of three housekeeping genes coding for cell division protein FtsH, thymidylate kinase, and IS 3 family transposase vary widely among phytoplasma species ( Guevara-Hernandez et al. Recently, the genome of ROLP was sequenced, and shares 99% and 98% similarity to the genome sequences of MBS and OY-M, respectively ( Zhu et al. Therefore, it is advantages to establish a rapid and accurate PCR system. The disadvantages of nested PCR are that it is time consuming, laborious, and provides nonspecific amplicons ( Demeuse et al. The assay involves two rounds of amplifications by PCR using universal primers, followed by restriction fragment length polymorphism (RFLP) analysis that provides definitive phytoplasma identification ( Lee et al. In particular, nested PCR assays using universal primers (P1/P7, along with primer set R16F2nb/R16R2b) based on the conserved 16S rRNA gene have been widely adopted for detecting phytoplasmas ( Ahrens and Seemüller 1992 Gundersen and Lee 1996 Lee et al. Methods to detect phytoplasmas, such as microscopy technique, were replaced by polymerase chain reaction (PCR) assays in the 1990s ( Nejat and Vadamalai 2013). Thus, it is essential to detect infected plants and insect vectors for disease management ( Nejat and Vadamalai 2013). 2008), the phytoplasma infection is mainly controlled by reduction of the insect vector population with insecticide applications ( Weintraub and Beanland 2006). Since phytoplasmas cannot be cultured in vitro and are transmitted by insect vectors from plant to plant ( Hogenhout et al. ROLP is a member of the ‘ Candidatus Phytoplasma asteris’ (16SrI) group, which comprises a number of related phytoplasmas including onion yellows phytoplasma (OY-M), aster yellows witches’-broom phytoplasma (AY-WB), and maize bushy stunt phytoplasma (MBS) ( Zhu et al. Recently, the disease has reemerged in southern China ( He et al. Since then, there have been no reports of epidemic outbreaks of this disease. By the 1980s, the disease broke out in southern China, causing serious yield losses in rice production ( Zhang et al. Disease symptoms include yellow and orange streaks appearing from the leaf apex, followed by leaf orange and scorch and death of whole plants ( He et al. In China, it mainly occurs in rice-growing areas of Yunnan, Guangxi, Guangdong, and Hainan provinces ( Chen et al.
Rice orange leaf disease (ROLD), caused by rice orange leaf phytoplasma (ROLP), was first discovered in Thailand in 1960s, and then found in India, the Philippines, Malaysia, and other Asian countries ( Hibino et al. The results showed that the distribution areas and vector carrying rate of ROLD had gradually increased. This method was used to survey the geographic distribution of ROLD in southern China from 2016 to 2018. It also minimizes the false positive problem produced by nested PCR. This method is simple and rapid, and its sensitivity up to 10 pg/μl of total ROLP DNA. In this study, we developed a PCR assay using a set of primers designed based on the ROLP genome sequence to amplify house-keeping gene FtsH-1 in rice and leafhopper vector samples. Current nested polymerase chain reaction (nested PCR) method using phytoplasma universal primers is widely used to detect phytoplasmas however, it has shortcoming of inconvenience and inaccuracy, for it needs two round of PCR reactions and could produce false positive results due to nontarget amplification. Accurate detection of the pathogen is important for disease management. ROLD severely devastates rice production in Asia. Rice orange leaf disease (ROLD), caused by rice orange leaf phytoplasma (ROLP), is transmitted by leafhopper vectors Recilia dorsalis and Nephotettix cinticeps.
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