Investigating the Latency Period of Grapevine Red Blotch Virus in a Diseased Cabernet franc Vineyard Experiencing Secondary Spread

Results and Discussion

Thirty-six GRBV-negative sentinel vines were planted in June 2015, primarily in an area of a red blotch-diseased Cabernet franc vineyard, where diseased vines were aggregated and secondary spread was previously documented (Cieniewicz et al. 2017b) (Figure 1). Therefore, sentinel vines were at risk of becoming infected with GRBV following natural S. festinus-mediated inoculations. Using sentinel vines was paramount to estimate the latency period because their GRBV-negative status was confidently ascribed at planting. As may be expected, the GRBV-negative status of vines already established in the study vineyard (originally planted in 2008) was less certain because the GRBV status of the mother stocks from which the buds and cuttings were sourced for grafting was unknown. Additionally, established vines may be infected with GRBV but not exhibit disease symptoms (Flasco et al. 2023a, 2023c), thus confounding our study.

The first sentinel vine to test positive for GRBV by multiplex PCR was identified in October 2018, but this vine was not symptomatic until October 2019. This vine was growing in the eastern vineyard area experiencing pronounced secondary spread (Cieniewicz et al. 2017b). The fact that the first GRBV-infected sentinel vine was found three years post-planting is consistent with an inefficient GRBV transmission rate from winegrape to winegrape (Flasco et al. 2021, 2023a, Hoyle et al. 2022). Similarly, the first GRBV-infected sentinel vine displayed disease symptoms four years post-planting, one year after testing positive for GRBV, which is consistent with previous observations from controlled S. festinus releases in the vineyard (Flasco et al. 2023a). Following 2018, more sentinel vines tested positive for GRBV and expressed foliar symptoms (Table 1), with most infected vines identified within or near the eastern area of the vineyard, where secondary spread has been previously documented (Cieniewicz et al. 2017b) (Figure 2). By 2023, more than two-thirds (69%, 25/36) of the sentinel vines were infected with GRBV (as shown by PCR) and exhibited red blotch disease symptoms (Figure 3 and Table 1).

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Figure 2

Distribution of sentinel vines testing positive for grapevine red blotch virus (GRBV) via polymerase chain reaction in a red blotch-diseased Cabernet franc vineyard, in October 2023. Grapevines exhibiting red blotch disease symptoms (i.e., foliar red blotches) are shown in gray, sentinel vines testing positive for GRBV are shown in red, and uninfected sentinel vines are shown in blue.

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Figure 3

Sentinel vines infected with grapevine red blotch virus in a diseased Cabernet franc vineyard experiencing secondary spread. (A) The first symptomatic (i.e., foliar red blotches) sentinel vine surrounded by diseased vines in October 2019, shown by a white arrow; (B) two symptomatic sentinel vines surrounded by symptomatic vines in October 2022, indicated with white arrows; (C) an infected but asymptomatic sentinel vine surrounded by diseased vines in October 2021; (D) a symptomatic sentinel vine in October 2021; (E) a closeup of foliar red blotch symptoms displayed by the sentinel vine shown in (D); and (F) a symptomatic sentinel vine in October 2023.

If a sentinel vine tested positive for GRBV via PCR in October, it also tested positive the following June (Table 1). Additionally, if a sentinel vine displayed foliar disease symptoms (i.e., foliar red blotches), it remained symptomatic through the remainder of the study, although the level of reddening observed on individual leaves and throughout the canopy varied between seasons (Figure 3). This observation on variable disease symptoms is consistent with a recent report (Rohrs et al. 2023). Environmental factors and/or viticultural practices such as water imbalance and fertilizer use may contribute to this variation in disease symptoms (Copp and Levin 2021). Together, our work conducted from 2015 to 2023 identified three distinct patterns occurring between the first instance of a vine testing positive for GRBV by PCR and the first appearance of foliar disease symptoms. The first pattern applied to sentinel vines that tested GRBV positive in PCR and were also symptomatic for the first time in October (20%, 5/25), perhaps suggesting no latency. The second pattern applied to sentinel vines that tested GRBV positive in PCR for the first time in June and became symptomatic the following October (56%, 14/25), indicating a four-month latency period. The third pattern applied to sentinel vines that tested GRBV positive in PCR for the first time in October and became symptomatic only the following October (24%, 6/25), illustrating a 12-month latency period. It would be interesting to investigate whether similar trends are observed in other viticultural regions where climate conditions and viticultural practices differ from those in Napa County, CA.

The lack of apparent latency observed for some sentinel vines (20%, 5/25) is difficult to understand because it took months, or even years, for a vine to display disease symptoms following Agrobacterium tumefaciens-delivery of an infectious GRBV clone in the laboratory (Yepes et al. 2018). Similarly, vines that tested GRBV positive in PCR following S. festinus-mediated inoculations were not symptomatic 16-months postexposure to viruliferous insects (Flasco et al. 2023a). A plausible explanation for the apparent lack of latency is that the GRBV status of these sentinel vines may have been misdiagnosed in June, as previously reported (Setiono et al. 2018, DeShields and KC 2023). Furthermore, some GRBV-positive vines in June may have been overlooked from 2018 to 2020, and in 2023, when sampling was only conducted in October of these years. More work is needed to thoroughly address this apparent lack of latency. Similarly, although visual surveys were conducted in October when symptoms were readily apparent, additional refinements are needed to verify our estimated four-to-12-month latency period because red blotch disease symptom development varies between and within vineyard blocks (Rohrs et al. 2023), and GRBV detectability varies early in the growing season (Setiono et al. 2018, DeShields and KC 2023). Monthly or bimonthly PCR testing and monitoring of sentinel vines for disease symptoms of the virus may enable verification of this estimated latency period.

To determine the phylogenetic clade to which the GRBV isolates infecting the sentinel vines belonged, additional assays were performed. Results showed that most sentinel vines were infected with GRBV isolates of phylogenetic clade 2 (92%, 23/25) (versus those infected with a GRBV isolate of phylogenetic clade 1 [8%, 2/25]). The skewed occurrence of GRBV clade 2-infected vines coincides with the greater incidence of clade 2 infections in the Cabernet franc study vineyard, particularly in the area with an aggregation of diseased vines and previously documented secondary spread (Perry et al. 2016, Cieniewicz et al. 2017a, 2019). The aggregation of diseased vines was previously speculated to be due to the presence of GRBV in the rootstock of the planting material because it took four years for disease symptoms to be apparent after planting (Cieniewicz et al. 2017b, 2019). Furthermore, all aggregated vines were infected with GRBV isolates of phylogenetic clade 2, and their genomic sequence was identical (Cieniewicz et al. 2017b, 2019). The presence of the same GRBV isolate in different vines can only result from the vegetative propagation of a single source of infected material. Nevertheless, a few GRBV isolates of phylogenetic clade 1 were found in infected vines (Flasco et al. 2023c and this study) and in viruliferous S. festinus caught on sticky cards in the Cabernet franc study vineyard (Cieniewicz et al. 2018). Together, these data showed that the type and distribution of GRBV isolates in infected sentinel vines mirrored the composition and distribution of GRBV isolates in the Cabernet franc vineyard.

Because the sentinel vines were free of GRBV prior to and at planting, their infection in 2015 to 2023 can be assumed to be from S. festinus-mediated inoculations. However, exactly when sentinel vines were inoculated by viruliferous S. festinus remains unknown. Interestingly, previous controlled-insect release experiments revealed that vines inoculated by viruliferous S. festinus did not show symptoms the same year of inoculation, although PCR testing showed that some vines tested positive for GRBV in the inoculated leaf tissue, but not in the mature leaves, such as those used in this study (Flasco et al. 2023a). Similarly, vines that tested positive the first time at 12-months postinoculation via viruliferous S. festinus were not yet symptomatic at 16-months postinoculation (Flasco et al. 2023a). By analogy, when considering the sentinel vine that was first confirmed as positive via PCR in 2018 and symptomatic in 2019, it is reasonable to speculate that this vine could have been inoculated by viruliferous S. festinus in June or July of 2017, or even in June or July of 2016.

GRBV-infected grapevines are asymptomatic in June (Setiono et al. 2018, Kahl et al. 2021, DeShields and KC 2023, Flasco et al. 2023a, 2023c), however, such vines can act as virus reservoirs and contribute to secondary spread mediated by S. festinus (Flasco et al. 2023a). With the new understanding that GRBV can be detected in grapevines that may not show symptoms until later in the growing season, or until the following growing season, symptomless GRBV-infected vines may serve as hidden contributors to secondary spread by S. festinus for an extended time, and on a large scale. Factoring in the estimated incubation time may extend this timeline and complicate disease management. Such information is essential to model spread trajectories, fine tune red blotch disease management recommendations, and better understand disease epidemiology.