Journal of Taiwan Agricultural Research

Author:Jye-Yann Liao, Chung-Ci Hu, Chin-An Chang, and Ting-Chin Deng*

Abstract:

    A naturally infected pineapple (Ananas comosus) sample (Pa1) was collected from major pineapple-growing area in Sheto and Ershui, Changhua County, Central Taiwan.  The diseased plant showed typical symptoms of tip dieback, reddening and downward curling of leaf margins, and early stages of green bumps on leaves.  The fruits of pineapple were infested with mealybug (Dysmicoccus brevipes) and sooty mold.  By electron microscopic observation, immunosorbent electron microscopy, coat protein electrophoresis, Western blot analysis, and nucleotide sequencing, the virus was identified as an isolate of Pineapple mealybug wilt-associated virus-1 (PMWaV-1).  Electron microscopic observations of diseased tissues revealed the presence of flexuous-shaped virus particles, 1250-1300 × 12-15 nm in dimension.  The virus consisting of coat protein subunit with an estimated molecular weight of 27 kDa was found.  The 783 3’-terminal nucleotides of this virus were sequenced and compared with corresponding sequences of PMWaV-1 (Access No. AF414119) listed in public databases.  We found that this virus is most closely related to the PMWaV-1 with nucleotide and amino acid sequence similarities of 99.1% and 98.4%, respectively.  The coat protein (CP) gene was further cloned into the vector pET28b (+), then used to express proteins in Escherichia coli BL21 (DE3) and to produce specific antiserum for future diagnostic purposes.  In Western blot, the prepared antiserum reacted with the expressed protein of CP gene and PMWaV-1 antigens in infected pineapple plant.  This is the first report of detection and identification of PMWaV-1 associated with infected Ananas comosus in Taiwan. 

Key words:Pineapple mealybug wilt-associated virus-1 , CP gene, Expressed protein, Sequence comparison

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