Viện bảo vệ thực vật/Plant Protection Research Institute (PPRI) - www.ppri.org.vn

A. Uke; T. X. Hoat; M. V. Quan; N. V. Liem; M. Ugaki and K. T. Natsuaki

     Cassava, Manihot esculenta Crantz, is the third-largest source of food carbohydrates, after rice and maize, in the tropics. On the Indian subcontinent, production of cassava can be severely affected by the cassava mosaic disease (CMD) caused by two Begomovirus species (Geminiviridae), Indian cassava mosaic virus and Sri Lankan cassava mosaic virus (SLCMV). In Southeast Asia, however, CMD was not known until May 2015, when the first CMD outbreak was observed in Cambodia, and the causal agent was identified as SLCMV (Wang et al. 2016). Here, we report the first outbreak of CMD in Vietnam. In May 2017, cassava plants showing typical CMD symptoms, including chlorotic mosaic, leaf distortion, and stunted growth, were observed in the field in Tay Ninh, Vietnam. Two symptomatic and two nonsymptomatic cassava leaves as well as two whiteflies on symptomatic leaves were collected from each of four independent farmers’ fields. Three whiteflies were also collected from an adjacent field with no symptomatic plants. Total DNA was extracted from each of the cassava leaves and individual whiteflies as described by Saghai-Maroof et al. (1984) and Rosell et al. (1999), respectively. The leaf DNA was used as a template in rolling circle amplification (RCA) to detect circular DNA molecules using a TempliPhi Amplification Kit (GE Healthcare). RCA products were detected from all DNA samples extracted from symptomatic leaves, but not from those from nonsymptomatic leaves.
      When the products were digested with HindIII, a single band of approximately 2.7 kb in size was obtained. Cloning of the band and partial sequencing of at least six independent clones revealed that the band was a mixture of two distinct DNA species: one most similar to DNA A of SLCMV and the other to DNA B of the same virus. This indicated that no geminiviruses other than SLCMV were present in symptomatic leaves. Three sets of SLCMV DNA A- and B-specific primers covering each of the whole SLCMV genome components were used in polymerase chain reaction (PCR) with a high-fidelity DNA polymerase KOD -Plus- Neo (Toyobo), and generated products of expected sizes from DNA isolated from symptomatic leaves, and whiteflies collected from them, but not from those of nonsymptomatic leaves and whiteflies collected from a neighboring field with no symptomatic plants.
      These overlapping PCR products covering the whole viral genome were directly sequenced, and the viral sequences obtained from cassava plants and whiteflies in all four cassava fields were found identical to each other and were deposited in the GenBank database (DNA A, LC312131; DNA B, LC312130). The sequences of DNA A and B were both most closely related (99.8% identity) to those of the Cambodian isolate of SLCMV (GenBank nos. KT861468 and KT861469) that belong to the India strain, and the DNA A sequence was only 93.7% identical to SLCMV Sri Lanka strain (GenBank no. AJ314737). According to current species/strain demarcation criteria (DNA A identities of 91 and 94% are the demarcation threshold for begomoviruses belonging to different species and strains, respectively) (Brown et al. 2015), the virus identified in Vietnam is thus an isolate of SLCMV India strain. The PCR amplification and sequencing of mitochondrial cytochrome oxidase 1 gene (cox1) of the whiteflies revealed that they belong to the Asia II 1 species of Bemisia tabaci species complex, as in the case of CMD outbreak in Cambodia (Wang et al. 2016). This is the first report of CMD caused by SLCMV in Vietnam and the second report of CMD caused by SLCMV in Southeast Asia, which produces 28% of cassava globally. This is a timely alert that appropriate control measures should be implemented to control the spread of the disease.

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