DETECTION OF HLVd AND HSVd
VIROIDS IN HOP PLANTS
Banja Luka, 2012
Tine Pokorn, Sebastjan Radišek, Branka Javornik, Jernej Jakše
HOP – Humulus lupulus L.
• dioecious perennial plant
• a member of the Cannabaceae family
• crosses among all taxonomic varieties produce fertile offspring
• only the female plant is important for commercial use
• is important agricultural crop in Slovenia
Slovenia's traditional hop growing region is found in lower
Savinja Valley
METAGENOMICS
• is the study of microbial populations in a sample by analyzing the
nucleotide sequence content
• next generation of sequencing (NGS) technologies, employing
massive parallel sequencing platforms, such as Roche 454, ABI
SOLiD, Illumina, Ion Torrent and Helicos
• sequences obtained from a studied infected plant using these
massive parallel technologies should also include specific
sequences of present pathogen(s)
VIROIDS
• are the smallest pathogens known so far
• they have from 246 to 475 nucleotides, are single-stranded,
covalent circular, non-encapsulated
• replicate autonomously in the host plant, spread systematically and
can cause disease symptoms similar to virus diseases
• viroids are divided into two taxonomic families: Avsunviroidae
Pospiviroidae
VIROIDS – Hop latent viroid (HLVd)
• first it seemed that viroid did not cause any visible
symptoms that is way it is called Hop latent viroid
• HLVd can reduce yield and alpha acid content by up to 30%
• it is 256 nucleotides long
• the disease can easily proceed from infected to healthy plants
via a foliar or vascular wound via cutting tools or other
agrotechnical machinery
VIROIDS – Hop stunt viroid (HSVd)
• it has been prevalent in Japan since 1970
• it is 294-308 nucleotides long
• the first symptoms can be seen at the beginning of June
• the concentration of viroids also increases with increasing temperature
causing lagging of plants
• symptomatic plants did not always clearly show HSVd infection, so a
more in depth investigation of any other possible pathogen was
required
• 8 healthy and 8 infected plants were sampled at 14-day intervals
beginning of the growing period
• detection protocol of HLVd and HSVd viroids in hop plants:
PRIMERS HSVd or HLVd
……AATTCTCGAGTTGCCGCATG
GGCAAGCAAAGAAAAAA……
• we compared the DNA and RNA isolation and we did not observe any
differences between the isolation methods for amplification of the viroid
• DNA isolation is much cheaper than RNA isolation
• after RNA isolation formaldehyde agarose gel electrophoresis was
used for evaluation of the integrity of the RNA samples
• all plants were infected with HLVd
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• the arrow indicates the faint amplicon, the length of which
corresponds to the HSVd expected length
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• we found HLVd in all analysed hop samples, including young plants
• concentrations are highest in the cones
• its identity was also confirmed by sequencing
• HSVd was not easy to amplify. We were able to amplify a faint
fragment of around 300 bp in infected samples, but some of the
faint bands were also present in healthy plants
• sequencing of the fragment confirmed sequence identity with HSVd
Questions?