Results and discussion
- Top of page
- Materials and methods
- Results and discussion
To the best of our knowledge, this study is the first comparison of the reliability of WI for grapevine virus-associated diseases in three different climatic conditions. The results indicate that WI is not always reliable for the detection of grapevine diseases that have been associated with specific grapevine viruses. Several factors appear to affect the reliability of this diagnostic test including success of transmission and the climatic conditions in which the indicator plants are grown. In the screenhouse and in the field-based hot and cool climates, viruses could be detected in at least one-third of each of the indicator/virus combinations, except for the Kober 5BB/GVA combinations (Table 2). Symptoms were not observed for any virus/indicator combination in the screenhouse (Table 2). Rugose wood symptoms were only observed on the Rupestris St George/RSPaV combinations at the hot climate site (Table 2). No rugose wood symptoms were observed on the LN33/GVB combination at any site. Fleck symptoms were only observed on the Rupestris St George/GFkV combinations at the hot climate site (Table 2) and symptom expression of each of the Cabernet Franc/GLRaV combinations was variable between the hot and cold climate field sites and between years at each site (Table 2). Symptoms were not observed on any uninoculated indicator plant at each site.
This study suggests that climatic conditions can have a significant impact on disease expression in grapevines. For example, leafroll symptoms associated with GLRaV-1, GLRaV-3 and GLRaV-9 were observed as early as 6 months post-inoculation in some Cabernet Franc indicator plants at the cool climate site, but the same symptoms were not observed in the same treatments grown in the field at the hot climate site or in the screenhouse (Table 2). By April (autumn) 2008, leafroll symptoms associated with GLRaV-1, GLRaV-2, GLRaV-3 and GLRaV-9 were observed on all inoculated Cabernet Franc indicator plants at both the hot and cool climate sites (Table 2). The symptoms associated with GLRaV-3 and GLRaV-9, however, were generally milder on the inoculated indicator plants grown at the hot climate site in 2008. The GLRaV-3 inoculated Cabernet Franc indictor plants and the uninoculated indicator plant growing adjacent to these vines did not grow well at this site, and it may be that these indicator plants did not display strong leafroll symptoms because of the poor growth. The reason for the poor growth of these grapevines is unknown.
Leafroll symptoms were never observed in the screenhouse during the three-year trial for any indicator plant inoculated with GLRaV-1, GLRaV-2, GLRaV-3 or GLRaV-9 (Table 2). However, each virus was detected in at least one indicator plant grown in the screenhouse, and this suggests that leafroll symptom expression was masked or suppressed in the infected indicator plants in the screenhouse environment. Cabernet Franc grapevines maintained in the screenhouse did not grow vigorously, and leaves were often pale in colour, suggesting that they may have been stressed. It is possible that continued growth of grapevines in smaller pots contributed to the stress exhibited by the grapevines, which may have affected symptom expression, but pot size was chosen due to restrictions on space for this trial.
The differential expression of leafroll symptoms between the hot and cool climate sites may be due to environmental differences, such as temperature. A higher temperature, e.g. ≥32°C, can reduce virus replication in other crops (Kassanis and Bastow 1971, Dawson et al. 1978, Glasa et al. 2003) and 28/23°C day/night (maximum/minimum) temperature reduced symptom expression in grapevines (Tanaka 1988). On average, the mean maximum monthly temperature at the cool climate site (monthly averages: minimum 3.7−13.2°C; maximum 12.0−27.7°C) are 3–5°C cooler than the hot climate site (monthly average: minimum 3.8–15.8°C; maximum 14.6−31.8°C) throughout the year, and mean minimum temperature is 0–3°C cooler during the year with the largest difference for both minimum and maximum temperature being observed during the summer months when grapevines are actively growing. The higher temperature at the hot climate site during the first growing season (minimum and maximum temperature was 3°C higher on average than the cool climate site) may have reduced the rate of replication of the leafroll viruses compared with that at the cool climate site and resulted in a lack of symptom expression because of low titre and/or low virus movement. In the second year, leafroll symptoms were observed at both sites, and it is likely that virus titre had reached a high-enough number to induce symptoms at both sites. We have observed that it can take more than 12 months for viruses to move from the point of infection to the shoots and cordons and reach a high-enough titre to be detected (Constable et al. 2012). It may be that viruses must be located in the shoots in high-enough concentration to cause symptom expression and that GLRaVs did not reach the shoots of inoculated grapevines at the hot climate site until the following season and thus remained symptomless in autumn 2007.
By April (autumn) 2009, leafroll symptoms were observed on all inoculated Cabernet Franc indicator plants at the hot climate site (Table 2), although the symptoms on vines inoculated with GLRaV-3 were not strong. At the cool climate site, suspected leafroll symptoms were observed as early as January (summer) 2009 on a few basal leaves of two-thirds of the Cabernet Franc indicator plants inoculated with GLRaV-3 and one-third of the Cabernet Franc indicator plants inoculated with GLRaV-9. From February (late summer) 2009 onwards, however, no symptoms were observed at the cool climate site in the same indicator plants even though these plants had tested positive by RT-PCR for the GLRaV with which they had been inoculated and which had previously expressed leafroll symptoms. No leafroll symptoms were observed from February (late summer) 2009 at the cool climate site on any Cabernet Franc indicator plants inoculated with GLRaV-1 or GLRaV-2. The reason for this differential expression between the two sites is unknown. It is possible that environmental factors such as the sudden onset of periods of unusually high summer temperature experienced at the cool climate site during January and February 2009 (14 consecutive days of maximum temperature ranging from 29.4 to 44.8°C: 8/14 days ≥34.9°C; 4/14 days ≥41.9°C) could have contributed to the suppression of leafroll symptoms perhaps because of a reduction in virus titre associated with a temperature ‘shock’.
Assuming that GLRaV species were the cause of leafroll symptoms in this trial, the PCR results of this experiment suggested that PCR testing may also not have been a reliable indicator of the transmission of GLRaV viruses. Leafroll symptoms were observed on all indicator plants inoculated with GLRaV-1 and GLRaV-9 at both the hot and cool climate sites at some time during the course of the three-year experiment. Each indicator sampled was tested for viruses by RT-PCR in April (autumn) 2009 and November (spring) 2009, but virus was detected only in two-thirds of the indicator plants for each virus at each site (Table 2). It is possible that this was due to inhibitors that affected the efficiency of the PCR test to detect viruses or that the samples that were taken did not contain sufficient virus. This suggests that at times, WI can be more sensitive than RT-PCR tests. It is also possible that the symptoms observed were associated with other problems and misdiagnosed as leafroll symptoms.
Differential expression of symptoms associated with GFkV-A was observed between the three sites (Table 2). That is, symptoms were not observed in the screenhouse even though GFkV-A could be detected, and symptoms were only observed in November (spring) 2009 on one Rupestris St George indicator plant at the cool climate site while symptoms were observed at the hot climate site during November (spring) of 2008 and 2009 (Table 2). GFkV-associated symptoms are usually observed in spring but do not persist throughout the growing season possibly because of the onset of periods of high temperature in summer (Tanaka 1988). However, if cooler temperatures do favour fleck symptom expression, then it seems that temperature alone may not explain the lack of symptoms observed at the cool climate site in this study. The average temperature for the cool climate site is 19.3–24.7°C/6.4–9.3°C (day/night) during October–December (spring-early summer) compared with the hot climate site, where symptoms were observed and which is warmer and has an average day/night temperature of 24−29.6°C/8.3−13°C during the same period.
Previous research has shown that a higher temperature (28°C/23°C day/night) can reduce the expression of leafroll and fleck diseases in grapevines compared with that at a lower temperature (20°C day and 17°C night) in greenhouse conditions (Tanaka 1988). It was also shown that shading can reduce symptom expression of both diseases, which may explain why these diseases were not observed on indicator plants in the screenhouse. It is possible that lack of experience in the interpretation of symptoms, particularly for subtle fleck symptoms that were not interpreted as those associated with GFkV, could result in false-negative results.
Indicator plants inoculated with GFkV-B did not show symptoms in any year at any site, suggesting that GFkV-B may be symptomless on the Rupestris St George indicator plants in hot and cool climates and in screenhouse conditions. The candidate grapevine infected with GFkV-A and used for inoculation of the indicator plants was co-infected with RSPaV, while no other virus was detected in the GFkV-B-infected candidate grapevine used for inoculation. It is possible that GFkV virus strains require co-infection with another virus or specific strain of a virus, such as RSPaV, for clear symptom expression to occur.
The RT-PCR results indicate that some virus species, e.g. the GVA strains used in this study, are less efficiently transmitted to biological indicator plants compared with other viruses such as GLRaV-3 (Table 2). The RT-PCR results of the grafted indicator plants suggested that except in one instance in the hot climate, neither GVA-V nor GVA-G were transmitted from the GVA-infected candidate plants, and this would explain why stem grooving symptoms were not observed. Similar results were also obtained in another study that used the same GVA-G strain for inoculation of Chardonnay and Shiraz grapevines (Constable et al. 2012). Candidate buds of GVA-V- and GVA-G-infected grapevines did appear to take on most of the indicator plants. It is possible, however, that the candidate buds did not make a vascular connection and were held in place because of the development of host callus tissue around the wound. It is possible that the low rate of GVA transmission during this experiment was due to a hypersensitive defence response induced in the indicator plants by the presence of GVA in the candidate bud, which prevented the virus from moving into the indicator plants. This could result in a false-negative result for GVA by WI. The requirement of a ‘helper’ virus such as GLRaV-1 or GLRaV-3 for transmission GVA by mealybugs has been suggested (Engelbrecht and Kasdorf 1990, Zorloni et al. 2006, Hommay et al. 2008), and it is possible that the presence of a helper virus is also required for transmission of GVA through grafting. It is also possible that the GVA strain used in this study infected the inoculated grapevines but remained below a detectable level throughout the study.
GVB-JS and GVB-RSG (Table 1) were detected in some inoculated indicator plants by RT-PCR at each site, but corky bark symptoms were not observed in any of the GVB-infected LN33 plants, and no conclusions can be made about the impact of climate on the expression of this disease (Table 2). One LN33 indicator at the hot climate site that was inoculated and tested positive by RT-PCR for GVB-RSG had some suspected and subtle pitting symptoms (Table 2). This symptom, however, by definition, was not corky bark, which must have externally visible bark swelling and phloem proliferation (Golino 1993, Martelli 1993). It is possible that the symptom observed fits the definition of LN33 stem grooving in which pitting and grooving symptoms are observed once the bark is stripped back to reveal the wood cylinder (Golino 1993, Martelli 1993). However, the symptom observed in this trial was milder than that which has been published for LN33 stem grooving (Martelli 1993), and it may be that what was observed was not associated with any virus infection, but with some other physical damage to the inoculated vine. The association between LN33 stem grooving and viruses is unknown. The GVB-RSG-infected candidate grapevine using chip bud inoculation was also infected with RSPaV and GLRaV-2, and it is possible that these viruses may have contributed to the appearance of the pitting symptoms on the LN33 indicator plant. No symptoms were observed in any uninoculated LN33 indicator plant.
The results of RT-PCR using the RSP48/RSP49 primer pair for the detection of RSPaV in the Rupestris St George indicator plants prior to inoculation identified a common problem in WI; the woody indicator plants may be infected with a latent virus. This result was confirmed by the detection of RSPaV using the RSP48/RSP49 primer pair in the both the inoculated and uninoculated Rupestris St George indicator plants after inoculation with different RSPaV isolates (Table 2). None of the uninoculated Rupestris St George indicator plants displayed stem pitting symptoms supporting the hypotheses that these indicators were infected by a latent strain of RSPaV.
RSPaV was detected by RT-PCR using the Sy9F/Sy8R primer pair in 11/18 inoculated indicator plants but not in any of the uninoculated control indicator plants (Table 2). At the hot climate site, all of the indicators inoculated with both RSPaV isolates tested positive for RPSaV using the Sy9F/Sy8R primer pair, and these inoculated plants also displayed stem pitting symptoms when the bark was removed by boiling or autoclaving. Stem pitting symptoms were not observed on any uninoculated indicator plant at the hot climate site. Stem pitting symptoms were not observed on any inoculated or uninoculated Rupestris St George indicator plant at the cool climate site or in the screenhouse (Table 2). These results indicate that some of the RSPaV strains detected using only the RSP48/RSP49 primer pair and not the Sy9F/Sy8R primer pair, may not be associated with rugose wood diseases, particularly as the uninoculated controls did not show symptoms.
Based on the RT-PCR results of this study, it is possible that the Sy9F/Sy8R primer pair is better at discriminating between disease-associated strains of RSPaV than the RSP48/RSP49 primer pair as suggested by Habili et al. (2006). However, RSPaV symptoms were not observed in the Sy9F/Sy8R-positive indicator plants at the cool climate site or maintained in the screenhouse. It is therefore possible that this primer pair also detected strains of RSPaV that are not associated with disease, or symptoms did not develop on the indicator plants because of other factors that might influence disease expression such as different climates. Further work is required to understand what influences different strains of RSPaV have on disease expression in grapevines and how these strains might interact with each other. Improved information about the genetic differences between strains and the factors involved in inducing disease could assist in developing a better diagnostic tool for disease-associated strains of this virus.
Rugose wood symptoms, including stem grooving, corky bark and stem pitting on Kober 5BB, LN33 and Rupestris St George woody indicator plants inoculated with GVA, GVB and RSPaV were not obvious in the hot and cool climate sites or the screenhouse in any year by simply peeling back the bark of plants growing in the field or in pots. Symptoms were observed only once the trunks were boiled or autoclaved, and the bark was removed; this procedure is recommended for detection of rugose wood diseases. It has been observed that corky bark symptoms associated with some strains of GVB may be observed on the green and lignified tissue of LN33 indicators in screenhouse conditions during Australian PEQ within three to six months of graft inoculation without any boiling or peeling of the bark (Mark Whattam, pers. comm., 2012).
Woody indexing relies on the interpretation of symptoms to diagnose a disease and does not necessarily identify specific viruses. Under some conditions co-infection of grapevines by two or more viruses may have a significant impact on symptom expression. For example, vein banding symptoms are observed when a grapevine is co-infected with Grapevine fanleaf virus (GFLV) and grapevine yellow speckle viroid (Krake et al. 1999), and roditis leaf discolouration may be associated with a co-infection of GFLV and Carnation mottle virus (Avgelis and Rumbos 1991). It is possible that latent virus infection of the indicator plant or the candidate grapevine by viruses such as RSPaV are important in triggering a host response to other viruses so that symptoms are expressed in biological indicator plants. Conversely, the presence of latent viruses may act to ‘cross-protect’ against other virus infections and result in a false-negative result. Cross-protection of mild strains against severe strains to prevent disease is a strategy used in other crops, such as citrus inoculated with mild strains of Citrus tristeza virus (CTV) to prevent decline, stem pitting and chlorosis symptoms associated with infection by severe strains of CTV (Fulton 1986). More work is required to understand how a combination of viruses and virus strains influences disease expression in grapevines.
In conclusion, the results of this experiment show that viruses may not be reliably detected by WI. Factors that effect WI include the success of chip bud inoculation with regard to bud-take and transmission of the candidate virus from the candidate bud to the grapevine indicator. Environment also impacts on symptom expression, and a screenhouse environment may not be optimal for WI for some viruses, particularly GLRaVs. Variable expression of symptoms was observed with time at each trial site and between both trial sites; the GLRaV-inoculated indicator plants at the cool climate site displayed symptoms during the first two seasons, and GLRaV-inoculated indicator plants at the hot climate site displayed visual symptoms during the last two seasons. These variations may also be associated with fluctuations in the environment. The specific environmental factors that influence symptom expressions are unknown, but we hypothesize that temperature may have a significant role. It is also possible that shading and day length could influence symptom expression. Thus, based on this trial, it is recommended that field-based WI, in conjunction with RT-PCR, continue to be carried out for a minimum of three years in Australia.