Journal of Taiwan Agricultural Research

Author:Chin-Chih Chen*, Jyh-Nong Tsai, Mei-Ya Chen, Jia-Yi Liao, and Fen-Lang Chiang

Abstract:

Strawberry (Haemanthus multiflorus Martyn.) samples showed brown mosaic or symptomless were collected in Miaoli, Taiwan. Using the newly designed primer pair SMYCPu/SMYCPd specific to strawberry mild yellow edge virus (SMYEV), an expected 838-bp DNA fragment containing the full-length coat protein (CP) gene of SMYEV was amplified by reverse transcription-polymerase chain reaction (RT-PCR). The nucleotide (nt) sequence of the amplicon was sequenced to verify the identity of the virus. Besides, the primer pair SMY273u/SMY273d was designed for RT-PCR to detect
SMYEV in strawberry fields of Miaoli and Wufeng, Taichung, with a detection rate of 10.6%. On the other hand, the SMYEV CP gene with a length of 726 nt was cloned into the expression vector
pET28a(+), and transformed into Escherichia coli strain Rosetta (DE3) cells to express a fusion protein of SMYEV CP with a molecular weight of about 26.7 kDa. The fusion protein was purified and injected into a New Zealand white rabbit to produce a polyclonal antibody against SMYEV CP (#206). In western blotting assays, the self-produced #206 as well as a commercially available  olyclonal antibody (Bioreba Ltd., Reinach, Switzerland) can react positively with the E. coli-expressed SMYEV-CP fusion protein and the commercial positive control of SMYEV, and has no non-specific reaction to healthy strawberry tissue. However, due to the low virus concentration in strawberries, these two antibodies failed to detect SMYEV an indirect enzyme-linked immunosorbent assay (indirect ELISA). Here, we first report the molecular identification and incidence of SMYEV in domestic strawberries. The polyclonal antibody and the RT-PCR methods developed in this study can be applied to establish the capability of the detection of SYMEV in strawberry plants. The developed methods could be useful in strawberry propagation to produce SYMEV-free seedlings.

Key words:Strawberry mild yellow edge virus (SMYEV), Molecular identification, Polyclonal antibody, RT-PCR detection

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