The transmission from source plants infected with different combinations of mixed variant infections was studied with the aim to understand the biological differences between GLRaV-3 variants responsible for leafroll spread in vineyards.
Grapevine leafroll-associated virus-3 (genus Ampelovirus; family Closteroviridae) is prevalent and economically important world-wide and associated with grapevine leafroll disease (GLD).
Phylogenetic studies up to 2018, have grouped GLRaV-3 isolates into nine different monophyletic groups and four supergroups, making GLRaV-3 a genetically highly diverse virus species.
In addition, new divergent variants have been discovered from around the world. In South Africa, results from a previous survey study showed that grapevine leafroll associated virus-3 (GLRaV-3) variants of group II and VI occurred predominantly (refer to Winetech-funded project GenUS11-1; Jooste et al., 2015).
This is an indication that these two variants of the virus might be more easily transmitted by the mealybug vectors, or that a combination of these variants in a plant can cause a more aggressive spread of the virus.
A transmission experiment using Planococcus ficus, the mealybug associated with GLRaV-3 spread, was carried out using source plants that were infected with a mixture of GLRaV-3 variants and viruses (Table 1).
A 24-hour Inoculation Access Period (IAP) and Acquisition Access Period (AAP) were used in the experiment where 20 first-instar nymphs fed on infected source plants (IAP) and the first-instar nymphs were then transmitted to healthy ‘Cabernet Franc’ recipient plants (AAP). A RT-PCR HRM analysis (Bester et al., 2012) was used to detect the transmission frequency of individual GLRaV-3 variants from the mixed variant infections in recipient plants after transmission.
Detection of the presence of other grapevine infecting viruses i.e., grapevine virus A (GVA), grapevine virus B (GVB) and grapevine rupestris stem pitting virus (GRSPaV), was carried out using virus-specific primers.
Results and discussion
Figure 1 provides illustrations of the outcomes from the RT-PCR HRM analysis which allowed for the determination of the GLRaV-3 variant groups. As can be seen from Figure 1A, there was an overlap in the melting curves of group I and II variants and this was resolved by undertaking a second analysis using a primer set that clearly separated these groups (Figure 1B).
In the study, 155 mealybug transmissions were carried out, 20 first-instar nymphs were transmitted to recipient plants after feeding on infected source plants. The success rate of the transmissions was above 80% for five of the source plants as a result of using 20 first-instar nymphs to induce a higher vector pressure.
The following results were obtained from plants infected with a mixture of GLRaV-3 variants and other viruses:
- Plant 20/7/2: The source plant was infected with variants I, II and VI; the group II variant was transmitted at a higher frequency, as well as the combination of group I + II variants. GVA was transmitted to 60% of the plants, GVE was not transmitted.
- Plant 13/2/19: The source plant was infected with group II and VI variants; again the group II variant was transmitted more efficiently. GRSPaV was transmitted to 5% of the plants.
- Plant 14/12/4: The source plant was infected with group I and III variants; the group I variant was slightly more readily transmitted, but the combination of variants I + III was transmitted most efficiently. GVE was not transmitted.
- Plant 8/7/1: The source plant was infected with group II and III variants; the group II variant was transmitted more efficiently than the group III variant. GVA was transmitted to 22% of the plants.
- Plant 3/17/16: The source plant was infected with group II and VI variants; the group II variant was transmitted most efficiently.
- Plant 10/20/6: The source plant was infected with group I and II variants; the combination of group I + II variants was transmitted slightly more efficiently.
- Plant 26/22/18: The source plant was infected with group I and VI variants; the group I variant was transmitted most efficiently. GVE was transmitted to 18% of the plants.
The transmission results indicated that variants I and II were transmitted more frequently to recipient plants either as a single transmission or in combination with each other.
The group VI variant was not transmitted as effectively as the group I and II variants in this study. In a similar study, the transmission dynamics of variants from group I and VI occurring in the Napa Valley in the USA, were examined by Blaisdell et al. (2012).
These authors concluded that vector transmission of the group VI variant alone was more frequent, followed by transmission with mixed infections of the two, while transmission with the group I variant alone was the least common. This was the first evidence that GLRaV-3 variants are biologically distinct.
The result of the group VI variant being transmitted more frequently in the USA study was not confirmed in the South African study. A possible explanation could be the more frequent presence of the group VI variant in the USA vines, different genotypes studied or interactions with other viruses.
Results from a previous survey study showed that grapevine leafroll-associated virus-3 (GLRaV-3) variants of group II and VI occurred predominantly in South African vineyards (refer to Winetech-funded project GenUS11-1; Jooste et al., 2015).
A reduced frequency of transmission of a single variant in mixed transmissions was observed, possibly due to competition between variants. In two of the source plants (14/12/4 and 10/20/6), a combination of variants, I + III and I + II, respectively, were transmitted more effectively than the single variants. In a previous study (PPRI 11/18), the four GLRaV-3 variants, representing group I, II, III and VI, in single infected vines, or when occurring in combination with GVA, were transmitted equally well under controlled conditions.
Statistically, no indication of variant-vector specificity was detected in the transmission experiments under controlled conditions. However, the Vitivirus GVA, was transmitted at higher frequencies from GLRaV-3/GVA infected plants and was transmitted without the simultaneous transmission of GLRaV-3.
The study demonstrated that GLRaV-3 variants from group I and II established at a higher frequency in recipient plants during transmission either as single transmissions or in combination with each other.
The exact interaction between variants in a plant is still unknown, but it is clear that competition between variants exists, either in the mealybug vector or in the recipient plant after transmission. The interaction between GVA and the group II variant needs to be investigated in more detail.
Not all grapevine farmers have the financial means to remove and replace infected vines and therefore the problem of leafroll will continue to have a serious economic impact in certain grape-producing regions.
This study aimed to support the management strategies of the industry through gaining knowledge of GLRaV-3 variants and other grapevine infecting viruses and their frequency of transmission.
The study concludes that complex interactions between the mealybug vector, the viruses themselves and grapevine exist and establishing vineyards with certified material and maintaining frequent scouting initiatives for the mealybug vector will assist to manage the disease.
The use of newer technologies, like next generation sequencing, can provide more in-depth answers to the pathogen status of a vine and this will assist the industry to ensure that all certified material is disease-free.
Bester, R., Jooste, A. E. C., Maree, H. J., and Burger, J. T. (2012). Real-time RT-PCR high-resolution melting curve analysis and multiplex RT-PCR to detect and differentiate grapevine leafroll-associated associated virus 3 variant groups I, II, III and VI. Virology J. 9, 219.
Blaisdell, G. K., Zhang, S., Daane, K., and Almeida, R.P.P. (2012). Patterns of virus transmission from hosts with mixed infections. Proceedings of the 17th Congress of the International Council for the Study of Virus and Virus-like Diseases of Grapevine (ICVG), Davis, California, USA, October 7-14, 2012: 178-179.
Jooste, A.E.C., Molenaar, N. Maree, H.J., Bester, R, Morey, L., de Koker, W.C., and Burger, J.B. (2015). Identification and distribution of multiple virus infections in Grapevine leafroll diseased vineyards. European Journal of Plant Pathology, 142 (2):363-375.
For more information, contact Elize Jooste at email@example.com