Virology

Expert Work

• Detection and identification of plant viruses and viroids
• Official monitoring of viruses and viroids
  • PPV
  • ToRSV in nepoviruses
  • RBDV
  • GPGV
  • PSTVd
• Monitoring of selected economically important viruses
  • grapevine viruses (Raspberry bushy dwarf virus (RBDV) and Grapevine Pinot gris virus (GPGV) on grapevine)
  • Pome and stone fruit viruses
  • WDV and YDVs
• Analysis of virus infections for the requirements of the certification body
  • grapevine
  • fruit trees
  • potato
• Preparing risk assessments
• Preparing expert opinions

Research work

• Identification and characterization of new viruses
• Epidemiology of viruses and viroids
• Research of small fruit viruses
• Studies of plant-virus-vectior interactions
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• Mechanisms of necrosis development on potato tubers after infection with PVY
• Searching for sources of resistance to virus infections (PPV)
• Research of plant physiology related to virus infections

Monitoring of Viruses and Viroids

Sharka caused by the Plum pox virus (PPV) is economically the most important virus disease of stone fruits. In very sensitive varieties, the PPV infection can cause complete yield loss and deterioration of trees, and in less sensitive varieties it primarily affects the quantity and quality of the fruit. PPV is a quarantine organism listed in Annex II.A.2 of Directive 2000/29/EC.  In Slovenia, the Plum pox virus has been under systematic survey since 1998. Since 2000, we have been focusing primarily on the production of  propagating material, namely the inspection of buffer zones of nurseries and orchards for graftwood production,  as well as endangered areas and obligatory testing of  nursery stock and mother trees of host plants.  We also pay special attention to testing the propagating material of stone fruits upon import or entry into the territory of Slovenia. The basis for performing the systematic supervision are the Rules on Measures for the Prevention and Suppression of Sharka, caused by the Plum pox potyvirus (Official Gazette of the RS, nos.18/02, 48/04, 68/05, 30/07 and 83/09). These Rules accurately prescribe the obligations and responsibilities of producers and other owners of host plants, supervisory bodies and expert institutions. The measures are designed so that they protect and preserve local (Slovenian) tree production and at the same time with a very high level of reliability ensure the production of non-infected propagation material and thus also protect the consumer, i.e. the customer.

Results of the systematic survey and control of Plum pox potyvirus in Slovenia

Systematic Supervision of Plum Pox in Slovenia from 1998 - 2005

Potato spindle tuber viroid (PSTVd) is a quarantine organism listed in Annex  I.A.I of Directive 2000/29/EC. In 2006, its presence was confirmed in the Netherlands on ornamental plants from the genus Brugmansia and from  the genus Solanum; this was the first finding of the PSTVd infection on these ornamental plants. In 2006 and 2007, infected plants from the Solanum and/or Brugmansia genera were also found in some other countries of the European Union. In 2007, the Dutch reported finding of PSTVd on Streptosolen jamesonii and the French on plants from the Petunia, Capsicum and Pseudocapsicum genera. Later, PSTVd infections were also confirmed on Physalis peruviana, Calibrachoa sp., Datura sp., Cestrum spp. and Dahlia sp. In Slovenia, the first PSTVd infection was confirmed on 27 October 2006 on the ornamental plant Brugmansia (Datura) suaveolens, which originated from a Dutch nursery. PSTVd is very dangerous to potatoes and tomatoes, since it can halve the yield on both hosts and it can be very quickly transmitted by touch and tools from an infected plant on to a healthy one. The Commission of the European Communities thus on 12 June 2007 adopted the “Commission Decision on Measures to Prevent the Introduction into and the Spread within the Community of Potato Spindle Tuber Viroid”, which also prescribes the survey on the presence or permanent absence of PSTVd in EU member states. In Slovenia a survey was started already at the end of  2006.  In the frame of the survey, in 2010 we confirmed the infection with CEVd (Citrus exocortis viroid) on S. jasminoides and the infection with TCDVd (Tomato chlorotic dwarf viroid) on petunias. These two viroids and also other representatives from the Pospiviroid genus, with the exception of IrVd 1 (Iresine viroid 1), can cause heavy losses in the production of important agricultural plants, therefore at the start of 2011 we prepared a risk assessment for all representatives of the Pospiviroid genus. It served as the basis for preparing a decision that prescribes the measures for preventing the introduction and spread of viroids from the Pospiviroid genus in Slovenia. Issuing the decision was necessary, because except for PSTVd and CSVd (Chrysanthemum stunt viroid), the pospiviroids are not classified as quarantine organisms in the European Community. CSVd is included on the list II.A.2 of Directive 2000/29/EC and is a quarantine harmful organism only for plants from the genus Dendranthema (Chrysanthemum) for planting, except for seeds.

The thermodynamic forecast of secondary structure of PSTVd nucleotide sequence  confirmed on pepino (S. muricatum) in Slovenia (NCBI GenBank label: HQ454932)

Determining and Characterising Viruses and Viroids

Viruses and viroids are the smallest organisms that cause plant disease. They are so small they cannot be seen with an optical microscope but can cause heavy losses of yield quantity and quality and even complete deterioration of plants. Some viruses and viroids do not cause outwardly apparent symptoms  of infection on certain host plants  and are therefore described as latent viruses and viroids Latent or mild infections are not detected by producers or are attributed to other causes but ccan have a negative effect on the agronomically important plant characteristics. Such plants  are also the source of infection for sensitive host plants.

The  species of virus and viroid can not be determined based on ,disease symptoms, therefore various laboratory techniques are necessary for their identification and detection. At the virology laboratory, we use traditional techniques such as mechanical inoculation of test plants, serological and molecular methods and electron microscopy. We develop and verify the effectiveness of new methods such as real-time RT-PCR , LAMP, etc.  We cooperate with companies and research institutes involved in development of new methods, have our presence in the field and cooperate with the extension service.

We have introduced and developed numerous new methods for detection of different important viruses and viroids.

Total RNA extraction method and Prunus species influence the detection of Plum pox potyvirus by real-time RT-PCR

Development and inter-laboratory evaluation of real-time PCR assays for the detection of pospiviroids

We were the first to confirm the presence of numerous viruses and viroids for the first time in Slovenia. Among the most important achievements is the discovery of the Raspberry bushy dwarf virus (RBDV) on grapevine, the only natural host outside the genus Rubus .

Studying the Epidemiology of Viruses and Viroids

We study the prevalence of various viruses, their variants and viroids in the region of Slovenia and also in other countries as part of international and bilateral projects. At the same time, we study the variability of virus populations in Slovenia and the spread of viruses with vectors such as scale insects, nematodes, bumblebees, etc., and we discover new host species.

VARIABILITY OF POTATO SPINDLE TUBER VIROID ISOLATES FROM ORNAMENTAL HOSTS IN SLOVENIA

DIVERSITY OF SLOVENE PPV (PLUM POX VIRUS) ISOLATES

SCALE insects - GRAPEVINE VIRUS VECTORS IN THE PRIMORSKA REGION

As part of the basic research project, during the 2000-2004 period we collected and analysed 811 samples from plant species belonging to 73 genera outside the genus Prunus in infected stone fruit plantations. We checked the results of serological testing with molecular techniques (RT-PCR) and confirmed infection in new host plants:  Covolvulus arvensis and Viola sp. Mazyad et al. (1992) confirmed the presence of the Plum pox virus in Solanum nigrum via mechanical inoculation, but we were the first to confirm the PPV infection in the plant in natural conditions (Viršček Marn et al., 2005).

Studying the Interaction Between Host Plants, Viruses or Viroids and Vectors

As part of the international project EUPHESCO, we tested the transmission of Potato spindle tuber viroid (PSTVd) via tomato roots and with bumblebees from PSTVd infected petunias to a non-infected tomato. In both trials, the transmission was not confirmed.

No transmission of Potato spindle tuber viroid shown in experiments with thrips (Frankliniella occidentalis, Thrips tabaci), honey bees (Apis mellifera) and bumblebees (Bombus terrestris)

Mechanical transmission of Potato spindle tuber viroid between plants of Brugmansia suaveoles and Solanum jasminoides and potatoes and tomatoes 

Studying the Effect of Viruses and Viroids on host Plants from the Agricultural, Genetic and Biologic Aspect

We engage in determining the effect of virus and viroid infections on host plants. As part of the research project ‘Studies of mechanisms in development of tuber necrosis due to PVY infection’, we determined the effect  of storage temperature and physiological age of tubers on the development of tuber necrosis after infection with PVYNTN.  High storage temperatures facilitated the development of tuber necroses. Prolonged storage at low temperatures caused a delay in the formation of tuber necroses and decreased the number of tubers with necroses. If the tubers were stored at low temperatures for at least 21 weeks, no necroses developed even after additional storage at higher temperatures.

In 2001 and 2002, we studied the effect of Plum pox virus (PPV) infection on the yield of peaches and determined that the yield decreased by 17% on average.

We are also interested in the infection’s effect on alteration in cells and the expression of genes.

Project presentation: Microarray studies of mechanisms in development of necrosis on potato tubers due to potato virus Y infection

Preparing Risk Assessments

At the beginning of 2011, we prepared a risk assessment for all viroids from the genus Pospiviroid for Slovenia. We assessed that latent infections of ornamental plants, that in Slovenia were frequently found to be infected with pospiviroids, potentially represent a great danger for Slovenian agricultural production. The loss of yield that would be caused by the infection with PSTVd and other pospiviroids on potato, tomato and sweet pepper in Slovenia would represent a serious threat to the economics of agricultural production on numerous farms and besides economic effects would also have social and environmental consequences.

Preparing Solutions for Issues in Agriculture that Arise Due to Virus and Viroid Infections

Our most important task is to prevent the spread of viruses and viroids and of the damage they cause. Thus, for example, as part of the CRP project “Solving problems of quarantine diseases for protection of stone fruit (Prunus spp) production”, we prepared a method for evaluating the immunity, resistance or tolerance of stone fruit varieties to PPV in Slovenia. Simultaneously, we also looked for sources of resistance or tolerance to PPV in the population of ‘Brkinska češpa’, a local biotype of ‘Požegača’.  As part of the second project, we determined that storing potato for 21 weeks at 4°C prevents development of necroses on potato tubers after infection with PVYNTN which significantly reduces potato tuber quality.