Development of a multiplex reverse transcription-PCR assay for simultaneous detection of garlic viruses

A preliminary screening for garlic viruses in garlic plants in Hunan,China,using existing monoplex（simplex） reverse transcription-polymerase chain reaction（RT-PCR） procedures detected four viruses/virus groups.These viruses/virus groups were Onion yellow dwarf virus（OYDV）,Leek yellow stripe virus（LYSV）,Shallot latent virus（SLV） and allexiviruses（e.g.,garlic viruses A,B,C,D,E,X）.Sequence analysis of the projected allexivirus amplicons revealed the allexivirus in the infected garlic plants was Garlic virus D（GarV-D）,which shared 92-97%sequence identities with various isolates from the world.A multiplex RT-PCR（mRT-PCR） was therefore developed to simultaneously detect and differentiate the four viruses/virus groups.To achieve this,four primer pairs targeting allexiviruses,OYDV,LYSV and SLV were designed.The anticipated amplicon sizes are 183 bp（allexiviruses）,265 bp（OYDV）,404 bp（LYSV） and 592 bp（SLV）,respectively.All primer pairs produced virus-specific fragments in both simplex and multiplex formats,thus confirming the efficacy of the newly developed mRT-PCR for detection of these viruses.The mRT-PCR further was evaluated by applying it to garlic plant samples collected in two geographic locations in Hunan.Allexiviruses,OYDV,LYSV and SLV were detected in 50.9,40.3,28.3 and 58.5%of leaf samples,respectively;and mixed infections with two or more viruses accounted for 54%of the garlic samples.The results obtained by mRT-PCR were confirmed by simplex RT-PCR assays.In conclusion,this newly developed mRT-PCR provides a rapid,sensitive and reliable method for the detection and identification of major garlic viruses.

Ethylene is Involved but Plays a Limited Role in Tomato Chlorotic Dwarf Viroid-Induced Symptom Development in Tomato

To investigate whether ethylene is involved in tomato plants in response to the invasion of viroid, tomato cv. Pearson and its ethylene-insensitive mutant Never ripe （Nr） was challenged with Tomato chlorotic dwarf viroid （TCDVd）. Both Pearson and Nr plants developed various symptoms including dwarf, bunch, reduced leaf size, leaf chlorosis, and necrosis at 3-wk-post-inoculation and thereafter even though the stunting in Nr was not as severe as in Pearson plants. The sizes of fruits were also significantly reduced in the viroid-infected plants. Treatments of Pearson with ethephon, an ethylene-releasing compound, mimicked TCDVd in induction of stunting and pathogenesis-related （PR） genes in the plants. Pretreatment of Pearson plants prior to TCDVd-inoculation or ethephon application with silver thiosulfate （STS）, an ethylene action inhibitor, partially suppressed the viroid- or ethephon-induced symptoms as well as PR gene expression. In addition, Nr and STS-treated Pearson exhibited a slightly lower viroid titre in comparison to the control Pearson plants. These results suggest that ethylene is involved but plays a limited role in TCDVd-induced symptom development.