Substantial input of two South African laboratories to worldwide knowledge.
To date, 70 virus species that can infect grapevines have been recorded.1 Two groups of viruses are especially detrimental for the grapevine industry from an economic point of view. These are viruses associated with grapevine leafroll (GLRD) and rugose wood diseases (GRWD). GLRD causes degeneration of phloem cells, which prevents translocation of synthesized carbohydrates from grapevine leaves. The disease delays maturation, decreases the sugar content of berries and, ultimately, negatively influences the quality of produced wine. In GRWD-affected plants, abnormal activity of cambium cells affects graft takes of cultivars to rootstocks. This leads to reduced vigour of grapevines and, ultimately, results in lower productivity and longevity of vineyards. The diseases are easily transmitted by pests, like mealybugs and scale, which are common in vineyards. Research has revealed many virus species of the families Closteroviridae and Betaflexiviridae1 associated with these two diseases, respectively. Each species is extensively genetically heterogenic with genome differences of up to 30% between genetic variants. The genome divergence sometimes correlates with putatively different pathogenicity to grapevines. The genetic and biological data obtained for these viruses is crucial for the reliable detection of these pathogens using molecular methods, like RT-PCR, and for the ongoing study of the aetiology of these diseases.
The current scientific data on viruses associated with GLRD and GRWD is the result of many years of study in various laboratories worldwide. It all began 41 years ago, in 1979, when Namba et al.2 in Japan revealed the consistent presence of flexuous virus particles in GLRD-affected grapevines. This finding was quickly confirmed by a laboratory in Switzerland. The presence of serologically different viruses, named Grapevine leafroll associated viruses, GLRaV-I and-II was detected.3 Later, the serologically distinct GLRaV-III, -IV and -V were identified by laboratories in the USA and France.4,5 Presently, the viruses are known as GLRaV-1, -2, -3, -4 and -5. Then, beginning in 1980, a few laboratories in Italy identified two viruses associated with GRWD, and named them Grapevine virus A (GVA) and B (GVB).6,7,8 All these new findings related to GLRD and GRWD attracted the attention of every laboratory working on grapevine viruses worldwide, including South Africa. At Plant Protection Research Institute (PPRI), which is now named Plant Health and Protection (PHP), of the Agricultural Research Council (ARC), equipped with a new electron microscope and highly trained staff in this field, all worldwide findings regarding different species of serologically distinct viruses were confirmed. Our laboratory, however, went one step further, and were among the first whose results suggested genetic and biological heterogeneity of virus species associated with GLRD and GRWD. In 1996 we mechanically transmitted GLRaV-2 to the alternative herbaceous host of this virus, Nicotiana benthamiana.9 Although GLRaV-2 was transmitted to this herbaceous plant earlier in Canada,10 our results suggested, for the first time, based on symptoms induced in N. benthamiana, the existence of the biological strains of this virus.9 The strains were sequenced at Cornell University, USA, and deposited in GenBank/EMBL database.11 Presently, it is well known that the virus has strains with different pathogenicity to grapevines. Six groups of divergent genetic variants were identified.12 One of them, the strain “Redglobe” (RG) is associated with inducing stem lesions/necrosis on various rootstocks after grafting.12
Analogous developments occurred with viruses GVA and GVB associated with GRWD. We confirmed that these viruses are transmissible to N. benthamiana. Moreover, our results revealed extensive genetic variability of GVA, which was correlated with different pathogenicity of variants to N. benthamiana.13 Also, the results strongly suggest that members of one of the molecular groups of variants of this virus, group II, are associated with Shiraz Disease (SD) (Figure 1A).14 The disease is highly destructive to noble grape cultivars Shiraz and Merlot in South Africa. Among eight full genome sequences of GVA deposited in GenBank, seven are from South Africa.15 In addition, we transmitted GVB to N. benthamiana and identified and fully sequenced 3 genetic variants of this virus.16 Four full genome sequences out of seven deposited in GenBank are from South Africa. Although the first paper that reported genetic heterogeneity of GVB originated in Australia,17 we were the first to suggest variants with different pathogenicity to grapevines. The virus is associated with grapevine corky bark disease (GCBD). The disease induces clear cane symptoms in the LN33 hybrid, which is used as an indicator of this disease in woody indexing of grapevines. In severe cases, the disease causes LN33 to “burst” between internodes (Figure 1B). We have GVB variants associated with severe CB symptoms in our collection, but we also identified a GVB variant that is present in LN33 which consistently and over years, does not exhibit any symptoms of this disease.18 The full genome sequence of this variant is deposited in GenBank. It is important to add that the ARC-PHP laboratory was the first to reveal the serological relation between GVA and GVB.19 These two viruses belong to the genus Vitivirus. Recently, after introducing a new sequencing technique called next generation of sequencing (NGS) or high through-put sequencing (HTS), the number of members of this genus rose to 11.20 Although investigation of these viruses has only just begun, it is possible that all members of this genus, as with GVA and GVB, are associated with GRWD. It means that they may be able to deregulate the differentiation of cambium cells, which is crucial for the graft take of grapevines.
FIGURE 1. Grapevine cv. Shiraz and LN33 infected with severe genetic variants of Grapevine virus A (GVA) and Grapevine virus B (GVB), respectively. The grapevines are also infected with GLRaV-3.
In South Africa, the main problem in vineyards is the widespread presence of GLRaV-3. An intensive study of this virus began in 2004 when the full (or nearly full) genome sequence of this virus was published by a laboratory at Cornell University, USA.21 In 2005, we used a technique called single strand confirmation polymorphism (SSCP) which, following the brief investigation of genetic heterogeneity of GLRaV-3 sequences, revealed two clearly divergent variants of this virus.22 Concurrently, an Italian group, also working on genetic heterogeneity of GLRaV-3 and using the same technique, published a paper on this subject.23 The results of our research were published the same year, although they only appeared in printed form slightly later.22 The ARC-PHP laboratory deposited the full genome sequences of three divergent genetic variants of GLRaV-3, named 621, 623 and PL20 in GenBank, representing I, II and III groups of this virus.24 Later, we identified a highly divergent genetic variant of GLRaV-3, which represented a new and yet unknown group VII of this virus.25 The full genome sequence of a variant of this group (GH24) was determined by the Vitis Laboratory, Stellenbosch University.26 The major contribution to worldwide knowledge on GLRaV-3 is credited to the aforementioned Stellenbosch laboratory. This includes the discovery that one of the terminal parts of the genome sequence, 5’ NTR, of this virus is 737 nt and not 158 nt as was previously published.27 The relevance of such a long genome “tail” remains unknown. It may play an important role in the biology of this virus. Moreover, the Stellenbosch University team is a leader in establishing the clarity of the genetic heterogeneity of GLRaV-3.26 The excellent work of this laboratory is reflected in many solid research and review papers, beginning in 2008.26,27,28,29,30 Currently, six groups of divergent genetic variants of this virus are known31 and this number may increase. However, despite this precise GLRaV-3 sequence data, nothing is known about the biological differences between variants of this virus.
In addition to GLRaV-1, -2 and -3, there is also GLRaV-4. The presence of a virus that is serologically different from GLRaV-1, -2 and -3 was noticed relatively early, in the USA, France and Switzerland.4,5,32 It was named as GLRaV-4, -5 and -6. Once virus genome sequence data became available from different laboratories, and the number of putative GLRaV species amounted to eight, it emerged that some of this data represented different genetic variants of a single virus species, which was named GLRaV-4.33 Long before the GLRaV-4 group was created we detected a virus named “band C closterovirus”, which cross-reacted with antibodies to GLRaV-6.34 The virus also cross-reacted with antisera to GLRaV-4 and -5 (Goszczynski & Kasdorf, not published). This suggested a serologically related group of viruses. Our finding was basically correct. We now know that the GLRaV-4, -5 and -6 are variants of one species, GLRaV-4. The exact identity of “band C closterovirus” remains unknown. As in the case of GLRaV-3, despite the clear genetic divergence between the presently identified eight variants of GLRaV-4, nothing is known about the differences in pathogenicity to grapevines of these variants.
Among viruses associated with GRWD there is also Grapevine rupestris stem pitting associated virus (GRSPaV). The virus has a different genome organisation from members of the genus Vitivirus, like GVA and GVB, and belongs to a separate genus, Foveavirus. GRSPaV was discovered almost concurrently in two American laboratories in 1998.35 Soon after the full genome sequence of GRSPaV was published, research data revealed that this mysterious virus is widely present in vineyards worldwide. Because it is commonly believed that this virus is not harmful to grapevines, we paid relatively little attention to it. At some point, however, an American laboratory discovered a highly divergent variant of this virus in grapevines affected by so-called Syrah decline (Sd).36 Although a high divergence between genetic variants of grapevine viruses is common, the media fuelled the suggestion that the newly-identified GRSPaV variant is associated with this disease. Sd severely affects graft of certain clones of cv. Syrah to rootstocks. Sd was identified in France and some French Syrah clones were imported to South Africa. Initially, a few mother blocks of these clones were established but, shortly after the report of Sd, propagation of these grapevines was stopped. An extensive survey conducted by the ARC-PHP revealed that none of the divergent genetic variants of GRSPaV is associated with Sd-affected grapevines in South Africa.37 Sd is now considered a genetic disorder and not associated with any grapevine pathogen. Currently eight genetic variants of GRSPaV are known, but the pathogenicity of variants to grapevines remains a mystery.35,38 Despite the fact that the virus is associated with grapevine rupestris stem pitting disease (GRSPD), the variants inducing symptoms of this disease in Rupestris St George grapevine have not been clearly identified.38
Genetic variability data is crucial for the precise detection of viruses. Currently, the most sensitive method of detection of grapevine viruses is RT-PCR. The technique requires a profound knowledge of targeted virus sequences to ensure reliable detection. Equally valuable as sequence data is biological data on grapevine viruses. We must bear in mind that from a strictly scientific perspective the viral aetiology of GLRD and GRWD has not been firmly confirmed. The third Koch’s postulate, which states that isolation of a pathogen and re-infection of a host with a pure culture of that pathogen, and which must be followed with the development of disease symptoms, has not been fulfilled. The reason for this lapse in knowledge is the fact that grapevines are usually infected with a mixture of various virus species and different genetic variants of a single virus species, and separation of them is not possible. Although some viruses, like GVA, GVB and GLRaV-2, were isolated in alternative herbaceous host N. benthamiana, transmission of them back to Vitis is impossible using current techniques. The only way to obtain a pure culture of grapevine viruses is to construct biologically active cDNA clones of these viruses in the laboratory.39 The cDNA clones for the following GLRD- and GRWD-associated viruses were constructed: GVA, GVB, GRSPaV, GLRaV-2 and -3.40,41,42,43,44 In the ARC-PHP laboratory, biologically active cDNA clones were constructed to GVA and GVB.44 Currently, the full data on virus infection of grapevines using cDNA clones has only been established for GLRaV-2 by a laboratory in the USA.43 Soon we can expect other research papers to confirm the successful infection of grapevines using cDNA clones of viruses. Recently an Italian laboratory published a paper on successful fulfilment of Koch’s third postulate for a member of the family Betaflexiviridae, Grapevine Pinot gris virus (GPGV), which is known to be associated with stunting, chlorotic mottling and leaf deformation of some grapevines.46
About 40 years of intensive research of grapevine leafroll (GLRD) and grapevine rugose wood (GRWD) diseases has revealed that many virus species of the two virus families, Closteroviridae and Betaflexiviridae, are associated with these diseases. Results have also revealed that each virus species is composed of a large number of divergent genetic variants, some of them with putative different pathogenicity to grapevines. Accumulated data is crucial for the accurate detection and further study of these important viruses. Two South African laboratories, ARC- Plant Health and Vitis Laboratory of Stellenbosch University, have played a significant role in creating this worldwide knowledge.
All the research described in this article was made possible by the financial support of Winetech, South Africa, and with the aid of institutions such as KWV, nurseries Vititec and Ernita, and with further help from people like J.H. Booysen, A. Andrag, R. Carstens, G. Kriel, N. Spreeth, T. Oosthuizen, J. Wiid and the late Prof. P. Goussard.
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– For more information, contact Dariusz Goszczynski at Plant Health and Protection, Agricultural Research Council – GoszczynskiD@arc.agric.za.