A potential threat to vineyards and an unexplored, fascinating subject for scientific study.

Grapevine species Vitis vinifera is planted on about 7.4 million hectares, being one of the most economically important crops worldwide. At the end of the nineteenth century, vineyards in Europe and South Africa were devastated by a tiny sap-sucking insect known as phylloxera (Dactulosphaira vitifoliae), which feeds on leaves and roots of grapevines. The insect was introduced to Europe from North America and was spreading fast in vineyards as it could not be controlled with chemicals. The grapevine industry was rescued with the introduction of grafting Vitis vinifera onto native American Vitis species, usually hybrids created from V. berlanderi, V. riparia and V. rupestris, which are naturally resistant to phylloxera. Since then grafting has become a common practice in viticulture.

Grafting, however, that combines two different Vitis species, is not without its problems. Perfect grafting requires full connection of vascular tissues (phloem and xylem) of grafted grapevine species. Failure of this connection, which may go unnoticed by the farmer, results in poor growth of grapevines, delayed bud burst, low fruit production, or decline of grafted plant after initial good growth. The problem with graft union compatibility is sometimes exhibited by external symptoms like “swelling” of scion above the graft union in some rootstock/V. vinifera combinations. Although the abnormally developed with graft unions can be environment-, rootstock- or scion-related, investigations revealed that virus infection of grapevines are also associated with these symptoms. The term “rugose wood diseases” (RWD) of grapevines refers to the modified grapevine woody cylinder (grooving and pitting), and exceedingly thick bark of a scion, which are putatively induced by virus infections.

The discovery of new viruses by researchers using the latest scientific diagnostic methods are inevitable. Although the grapevine industry cannot ignore these new findings, the importance of these emerging viruses must be confirmed by Koch’s postulates, or their etiological role must be confirmed by association with certain diseases. Both results will help to improve the phytosanitary status of our material.

A comprehensive progress review in the study of RWD, between 1962 to 2013, which provided a solid foundation for further investigation of this disease, was published in the Journal of Plant Pathology in 2014. Briefly, a breakthrough in the clear identification of RWD was the discovery of grapevines especially sensitive to RWD. This has led to the identification of four diseases of rugose wood complex: Rupestris stem pitting (RSPD), Kober stem grooving (KSGD), corky bark (CBD) and LN33 stem grooving (LNGD). The discovery was followed by descriptions of virus species associated with RWD, grapevine virus A (GVA) and grapevine virus B (GVB), and transmission of these viruses between grapevines by insect pests common in vineyards, such as mealybugs. The full molecular characterisation of genomes of these viruses has led to establishment of a new taxonomic group, the genus Vitivirus. In addition to the members of this group, viruses of the genus Foveavirus, like grapevine rupestris stem pitting-associated virus (GRSPaV), discovered in 1998, were also found to be associated with RWD. The number of recorded members of the genus Vitivirus, rapidly expanded after the introduction of high-through sequencing (HTS). This modern technique generates sequence data for any genetic material, including viruses, present in the investigated sample. Currently 11 virus species of the Vitivirus genus, named with sequential letters of the alphabet: GVA, GVB, GVD, GVE, GVF, GVG, GVH, GVI, GVJ, GVK and GVL, were discovered in grapevines in Italy, France, Japan, South Africa, New Zealand, USA, Argentina and South Korea. It indicates that the genus Vitivirus comprises a large number of virus species and suggests that these viruses are common in vineyards worldwide.

This is highly intriguing considering that accumulated research data strongly suggest that two of them, GVA and GVB, induce two diseases of RWD complex, KSGD and CBD, respectively. Thus, should we consider that the remaining nine vitiviruses, GVD – GVL, are also potentially able to cause abnormal development of graft unions? If yes, the industry should be concerned. There are reports that the vitiviruses, unlike members of the family Closteroviridae, associated with grapevine leafroll disease (GLRD), are relatively difficult to eliminate from grapevines using commonly used virus elimination techniques. In addition, these viruses are easily transmitted between grapevines by common vineyard pests like mealybugs.

There are many questions regarding vitiviruses that should be answered. If these viruses are really widely present in vineyards worldwide and are able to induce RWD, what prevents them from inducing pathological changes in graft unions? Perhaps only a small number of grapevine cultivars are susceptible to RWD? Or, perhaps only a small percentage of populations of genetic variants of each member of the Vitivirus genus are pathogenic to grapevines. It is possible that a high titre of vitiviruses is needed to visualise the pathogenic effect of these viruses in grapevines and to reach this hypothetical threshold titre they might need support from other viruses.

In South Africa, CBD-affected LN33 grapevines are always infected with GVB and a closterovirus, grapevine leafroll-associated virus 3 (GLRaV-3). This virus and the other members of the family Closteroviridae encode strong suppressors of anti-virus grapevine defence system. A recent article published by the USA laboratory strongly suggests that the synergy between grapevine vitiviruses and grapevine leafroll-associated viruses is a reality. It was found that the titres of GVA and GVB in co-infection with the closteroviruses, GLRaV-2 and GLRaV-3, were clearly higher than the titres were in the absence of these co-infections. In addition, despite the association of vitiviruses with RWD, the pathogenicity of these viruses to grapevines is still not well known. An anatomical study of CBD-affected grapevine revealed abnormal development of xylem and phloem tissues. Thus, GVB, which is the suspected cause of this disease, is able to deregulate differentiation of cambium cells to xylem and phloem. How does GVB do it? What grapevine genes are targeted? Does GVB, and other vitiviruses, have a special “affinity” for grapevine meristematic cells? All these questions can be answered only if we have established an easy model to study vitivirus-grapevine host interactions.

The ideal is the model comprising a LN33 hybrid grapevine (Courderc 1613 x Thompson Seedless) and GVB-associated with corky bark disease (CBD). Although the RWD-associated viruses do not induce easily visible external symptoms in most grapevine cultivars, CBD is an exception. LN33 reacts to CBD infection with severe swelling and longitudinal cracking of canes between internodes (Figure 1). The symptoms are the result of the rapid and excessive proliferation of the secondary phloem cells. After CBD infection of LN33 grapevine the symptoms of the disease appear consistently every year in new growing canes. This secures a constant supply of genetically identical CBD-affected grapevine tissues for study. Also, it was found that young, in vitro-propagated LN33 plants are very susceptible to CBD. Transmission of CBD by micro grafting induced clearly visible swelling of stems of LN33 plants in 8 – 12 weeks.

 

Canes of LN33, which are disease-free and

exhibit severe symptoms of CBD.

 

The second component of the GVB/LN33 model are cDNA clones of GVB variants. The construction of biologically active and stable cDNA clones of GVB is crucial. Although GVB was isolated from grapevines by mechanical transmission of this vitivirus to its alternative herbaceous hosts, Nicotiana sp., transmission of this virus back to grapevines is impossible with current knowledge. Our laboratory has made clear progress towards creating the model to investigate pathogenicity of GVB to LN33 grapevines. The virus is extensively genetically heterogenic. Currently seven complete genome sequences of genetic variants of GVB, sharing 74.9 – 85.2% nucleotide identity, are available in the GenBank/EMBL database. Four of these variants were identified in our laboratory. Also, we reported that the genetic variants of GVB differ in pathogenicity to LN33 grapevine. LN33 plants in our grapevine collection are infected with variant GVB-H1 and, over the years, consistently do not express CBD symptoms. The genome sequence of this variant, that is not pathogenic to LN33, is clearly divergent from other GVB variants. Finally, we constructed biologically viable cDNA clones of GVB. The clones are crucial for the pathogenicity study of GVB variants, because constructing of clones is the only way to obtain a pure culture of genetic variants of this grapevine virus. In South Africa, CBD-affected LN33 grapevines are usually infected with a mixture of various divergent genetic variants of GVB and other viruses, like GRSPaV and GLRaV-3. Separating GVB from mixed virus infections is practically impossible. The GVB/LN33 model has enormous scientific potential. The severe and relatively quick reaction of LN33 to GVB infection, the easy manipulations in the genome of GVB using cDNA clones of this virus, and the comparative analysis of expression of the virus and molecular changes in grapevine cells infected with GVB genetic variants differing in pathogenicity to grapevines, may reveal which part of the GVB genome confers CBD-inducing pathogenicity determinants of the virus, and how the virus de-regulates cambium cell differentiation. This could lead to identification of grapevine genes targeted by GVB in CBD disease development and modification of these genes to make grapevines not susceptible to GVB, and possibly also to other vitiviruses.

 

Acknowledgement

The author thanks Winetech, South Africa, for the financial support of this study.

 

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– For more information, contact Dariusz Goszczynski at GoszczynskiD@arc.agric.za.

 

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