JOURNAL FÜR KULTURPFLANZEN, 66 (7). S. 248–254, 2014, ISSN 1867-0911, DOI: 10.5073/JFK.2014.07.04 VERLAG EUGEN ULMER KG, STUTTGART Originalarbeit Angelika Ziegler1, Magdalena Kawka2, Marcin Przybys2, Teresa Doroszewska2, Urszula Skomra2, Ute Kastirr1, Jaroslav Matoušek3, Jörg Schubert4 Detection and molecular analysis of Hop latent virus and Hop latent viroid in hop samples from Poland Nachweis und molekulare Analyse des Hop latent virus und Hop latent viroids in polnischen Hopfenproben 248 Abstract zur Eliminierung von Viren und Viroiden im Hopfen gestar- tet. In den Jahren 2012/13 wurden in vitro Pflanzen, Pro- Monitoring the occurrence of virus diseases in plants is ben aus der IUNG-PIB Versuchsstation und aus kommer- important for the implementation of early control mea- ziellen polnischen Hopfengärten auf das Hop latent virus, sures and prevention of further disease spread. In Poland, Hop latent viroid und Hop stunt viroid getestet. Für die in 2004 a health programme for hop was started to elim- Virustestung wurden RT-PCR und ELISA eingesetzt. Die inate viruses and viroids. In 2012/13, in vitro plants, sam- Viroide wurden mittels RT-PCR nachgewiesen. Insgesamt ples from the IUNG-PIB experimental station and com- war die Nachweishäufigkeit für Viren und Viroide gerin- mercial hop gardens in Poland were tested for Hop latent ger als vor dem Start des Programms. Klonierung und virus (HpLV), and Hop latent and Hop stunt viroids (HpLVd Sequenzierung lassen den Schluss zu, dass das Hop latent and HpSVd). For virus testing, RT-PCR and ELISA meth- virus und das Hop latent viroid aus den polnischen Pro- ods were used. In order to detect hop viroids, RT-PCR was ben den „type“ Sequenzen und den tschechischen Viren/ employed. The overall incidence of HpLV and hop viroids Viroiden sehr ähnlich sind. was lower than reported before the start of the programme. Cloning and sequencing revealed that the HpLV and the Stichwörter: Hop latent virus, hop latent viroid, hop stunt HpLVd from Polish sources are very similar to the type viroid,RT-PCR,real time RT-PCR,ELISA sequences and the Czech sources. Key words: Hop latent virus, hop latent viroid, hop stunt Introduction viroid,RT-PCR,real time RT-PCR,ELISA Hop latent virus (HpLV) is a carlavirus infecting hop. The virus was first described by SCHMIDT (1966). There are no Zusammenfassung apparent symptoms; however, some impairment of growth and reduction in yield has been noted. HpLV is transmit- Die Überwachung von Viruskrankheiten bei Pflanzen ist ted by the aphid Phorodon humuli in a non-persistent man- wichtig für die Durchführung frühzeitiger Kontrollmaß- ner (ADAMS and BARBARA, 1982). Other aphids (eg M. persi- nahmen und die Verhinderung der weiteren Ausbreitung cae) and mechanical means (scissors, contact of bines) are der Erreger. In Polen wurde im Jahr 2004 ein Programm also implicated in HpLV transmission (CROWLE et al., 2006). Institute Institute for Epidemiology and Pathogen Diagnostics, Julius Kühn-Institut – Federal Research Centre for Cultivated Plants, Quedlinburg, Germany1 Institute of Soil Science and Plant Cultivation-State Research Institute, Department of Plant Breeding and Biotechnology, Pulawy, Poland2 Biol. Centre AS CR v.v.i., Institute of Plant Molecular Biology, 37005 České Budějovice, Czech Republic 3 Institute for Biosafety in Plant Biotechnology, Julius Kühn-Institut – Federal Research Centre for Cultivated Plants, Quedlin- burg, Germany4 Correspondence Dr. Angelika Ziegler, Julius Kühn-Institut, Institute for Epidemiology and Pathogen Diagnostics, Erwin-Baur-Str. 27, 06484 Quedlinburg, Germany, E-Mail: [email protected] Accepted 31 January 2014 ANGELIKA ZIEGLER et al., Detection and molecular analysis of Hop latent virus and Hop latent viroid ... Hop is the only known natural host. However, hemp can sions had not been tested for virus and viroid infections Originalarbeit serve as an experimental host for HpLV (ZIEGLER et al., so far. Samples were also collected from 8 commercial 2012). hop gardens located in different regions of Poland. These Hop latent viroid (HpLVd, genus Cocadviroid; PUCHTA et commercial hop gardens were established between 2005 al., 1988) is distributed worldwide and appears on hop and 2008 using disease free stocks of cultivars Sybilla, usually symptomless. However, a decrease in contents of Magnum, Iunga, and Lubelski produced at IUNG-PIB. alpha acids, important for brewing, has been reported for Testing included 136 samples from the hop collection infected plants (for example, PATZAK et al., 2001). and 74 samples from commercial hop gardens. From each Hop stunt viroid (HpSVd, genus Hostuviroid) has recently hop garden, 6–12 leaf samples had been collected, depend- been detected in hops for the first time in Europe, in Slove- ing on the size of the plantation. Samples were frozen at nia (RADISEK et al., 2012). The viroid severely stunts the –80°C immediately after collection. The in vitro plants plants, leads to smaller yields, loss in alpha acids (SANO, were from IUNG-PIB stockcollection of the most popular 2003) and changes in expression of important genes Polish hop cultivars. 18 samples of in vitro plants obtained involved in regulation of the metabolome (FÜSSY et al., using the method of regeneration of apical tips were tested, 2013). Typical symptoms also include leaf curling and small and 14 samples of plants obtained from sterilized seeds cones. Stunting appears 3 to 5 years after plants become germinated in LS medium.The source for the HpLV and infected (EASTWELL and NELSON, 2007). Just like HpLVd, HpLVd samples used for comparison was infected hop HpSVd can be transmitted by hands, scissors or through from the glasshouse. HpLV originated from the Czech bine contact. HpSVd has a wide host range which includes hop virus collection (provided by J. PATZAK). 249 woody plants (plum, peach: SANO et al., 1989; citrus: DIENER et al., 1988; pistachio: ELLEUCH et al., 2013) and grape- Virus purification, ELISA, Western blot vines (e.g. MATOUŠEK et al., 2003). These can serve as a Hemp (Cannabis sativa) is a useful propagation host for natural reservoir of viroid. There are no genetic resources HpLV. Unfortunately it could not be used for virus purifi- available for breeding for HpLV, HpLVd or HpSVd resis- cation as it contains a cryptic virus (ZIEGLER at al., 2012). tance, therefore growing healthy hop plants is important Consequently, we had to use HpLV infected hop grown in to prevent spread of these pathogens. the greenhouse. Before starting the purification, the mate- Since hop yards are kept for 20 years and longer, con- rial was tested by electron microscopy for the presence of tinuous monitoring is necessary to be able to remove any virus particles. Plant material (200 g leaves and shoots) infected plants as early as possible and to prevent spread was harvested and homogenized in a Warren blender with of the disease. 500 ml of ice cold 0.1 M potassium citrate/0.02 M EDTA Poland is one of the largest producers of hop. After it buffer pH 7.0 containing 1% (w/v) Polyclar AT. Plant debris became evident that Polish hop yards had a high inci- was removed by filtering through cheese cloth; the fil- dence of hop viruses and viroids (SOLARSKA and GRUDZINSKA, trate was centrifuged for 10 min at 20.000 g. The superna- 2001), a programme was started at the Institute of Soil Sci- tant was clarified with 0.1 volume tetrachloromethane. ence and Plant Cultivation-State Research Institute (IUNG- Virus particles were pelleted by centrifugation for 1 h PIB), Pulawy, to produce disease-free stocks. To estimate 30 min at 110.000 g in a vertical rotor. The pellet was the impact of the programme, in 2012/2013 samples from redissolved overnight in 20 ml potassium citrate buffer commercial hop gardens established using disease-free containing 0.5% Triton X 100. Aggregates were removed stocks and from experimental field “Kepa IUNG-PIB”, by centrifugation for 5 min at 20.000 g. The supernatant where genetic resources of hop from around the world was layered onto a sucrose cushion (40%) and centri- are maintained and could be a source of these pathogens, fuged at 98.000 g in a vertical rotor. The pellet was redis- were collected and analysed by RT-PCR. In addition, solved in 3 ml sodium phosphate buffer (0.1 M, pH7), using RT-PCR, the presence of HpLV, HpLVd and HpSVd loaded onto a preformed sucrose gradient (10–40%) and in the vegetatively propagated in vitro stock collection centrifuged at 110.000 g for 1 h 30 min. Two light scat- IUNG-PIB of most popular Polish hop cultivars and in hop tering bands were visible; the material from these 2 bands seedlings obtained from seeds was studied to establish was removed from the tubes, diluted with an equal vol- the health status of the plants. Additionally, at the Julius ume of sodium phosphate buffer and centrifuged for 1 h Kühn-Institut (JKI), Federal Research Centre for Culti- 20 min at 150.000 g. This resulted in pellets containing vated Plants, Quedlinburg, a polyclonal antiserum was highly purified virus, which was used for immunization produced for the detection of HpLV and tested for appli- of a rabbit using a common intravenous immunization cation in ELISA and Western blot. scheme. The rabbit was bled after 3.5 months and serum used for immunodetection. For coating of ELISA plates (Nunc) leaf extracts were Material and methods diluted 1:5 in PBS and kept overnight at 4°C. After 3 washes with PBSTw and blocking with 2% MPBSTw, the Plant material anti-HpLV antiserum was added (1:5.000 in MPBSTw) to Hop samples were collected from IUNG-PIB experimen- the wells and incubated for 3 hrs at room temperature. tal field “Kepa”, where various hop cultivars from all over An incubation with the secondary antibody (anti rabbit AP the world are maintained.