Identification And Molecular Characterization Of Pathogenic Viruses Infecting Taro

Taro (Colocasia esculenta) is a herbaceous plant in the genus Colocasia of family Araceae, which is widely cultivated as a staple food or vegetable in the world. Viral diseases seriously damage the production of taro. In this research, we investgated the occurrence dynamics of taro viruses in China. PCR and reverse transcription PCR (RT-PCR) were employed for the identification of pathogenic viruses in taro, aiming at finding out the species and situation of taro virus, and also providing technical support for building the detection and treatment system of taro virus. The research result as follows.1. Field investigation of viral diseases on the cultivated taro in the National Aquatic Vegetables Resources Nursery of Wuhan Vegetables Reserch Institute in Hubei province was conducted on September30th,2010. Results revealed that the viral diseases were widespread, most of which exhibited the symptoms of mottle-leaf, feathery mottle, deformity shrinks and chlorotic spots with the exact frequencies of26.92%,16.67%,28.21%and3.85%, respectively.2Reverse transcription PCR (RT-PCR) was employed for the detection of Dasheen mosaic virus (DsMV) in taro from Hubei, Zhejiang and Shandong provinces. Results revealed that the average viral infection frequency in129collected taro samples was27.9%. Ninety-nine field samples from Hubei province had the frequencies of9.5%-42.9%, ten field samples from Zhejiang Jinhua had the frequency of40.0%, three field samples from Shandong Linyi were all negative. These112field samples all had distinct virus symptoms. Seventeen tissue cultured samples of taro from Wuhan Vegetables Reserch Institute were symptomless and had the highest frequency of58.8%. RT-PCR products of317bp (covering partial coat protein gene) from14isolates were sequenced. Results showed that the obtained sequences had high intra-isolate nucleotide similarities, but inter-isolate nucleotide similarities were varied from68.3%to97.8%. The CP genes of two DsMV isolates (DsMV-SCS、DsMV-ZP) from Hubei province and one DsMV isolate from Zhejiang province named DsMV-JH were sequenced, and their sizes were951bp,942bp and987bp, respectively. Their CP genes shared similarities of78.2%,79.0%,85.5%at nucleotide (nt) level and85.0%,82.3%,89.2%at amino acid (aa) level to each other and similarities of73.0%-78.9%,73.9%-91.9%,74.3%-92.1%at nt level and77.0%-86.5%,74.8%-97.1%,74.8%-98.2%at aa level to reported CP sequences of DsMV, respectively. The phylogenetic trees constructed based on nucleotide and deduced amino acid sequences of the CP gene of DsMV showed that there was no obvious correlation between phylogenetic positions and host or geographical origins of different DsMV isolates3PCR was employed for the detection of Badnavirus in26taro samples with the Badnavirus universal primers (BadnaFP/BadnaRP), the target fragment of about600bp was obtained from13samples. The result of sequence analysis revealed that12clones form7samples had the conserved sequence of Badnavirus, and the sequences form other6samples did not had the conserved sequence of Badnavirus. Therefore, the Badnavirus universal primers (BadnaFP/BadnaRP) were not fit for detection. The Badnavirus sequence analysis of12chones from7samples showed that their intra-and inter-isolate similarities were89.9%-100%,78.8%-99.5%at nucleotide (nt) level and94.8%-100%,81.8%-99.5%at amino acid (aa) level, respectively. The obtained sequenes showed lower than80%similarity with the Badnavirus sequence in NCBI., the phylogenetic analysis showed that it was a new virus specie in Badnavirus. The new primers (P197/P433) were designed from the obtained Badnavirus sequences for detection of this virus. The result of detection on51taro samples showed that38samples had the target fragment of257bp.4. In order to clone the full length of this Badnavirus genome, primers of P197and RP1408were designed by aligning the obtained sequences.13clones of target sequences were obtained from8positive samples with the length of1724bp-1771bp. Their intra-and inter-isolate similarities were85.5%-98.9%and85.5%-99.6%at nucleotide (nt) level. Sequence analysis confirmed that about600bp of5’-terminal region of these sequences were conserved regions of RT and Rnase H gene of Badnavirus, but the1.1kb of3’-terminal region of these sequences were not the Badnavirus sequence. The primers of P1161and RP1408designed followed the1.1kb of3’-terminal region of these sequences could amplify target sequence from all of the samples (including the positive and negative taro samples of this virus and a cucumber leaf sample), which confirmed that the1.1kb sequence was the taro genome’s sequence. Furthermore, we extracted the taro genome of two positive samples from agarose gel. Target virus sequence were also obtained when these taro genomes were used as template to conduct PCR with the detection primers of P197and P433, this further illustrated that the Badnavirus sequence had been integrated into the taro genome, but the full length genome amplification of this Badnavirus was unsuccessful.