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Citation: Chooi, K.M.; Bell, V.A.;
Blouin, A.G.; Cohen, D.; Mundy, D.;
Henshall, W.; MacDiarmid, R.M.
Grapevine Leafroll-Associated Virus
3 Genotype Influences Foliar
Symptom Development in New
Zealand Vineyards. Viruses 2022,14,
1348. https://doi.org/10.3390/
v14071348
Academic Editors: Olivier Lemaire,
Etienne Herrbach and Feng Qu
Received: 11 April 2022
Accepted: 17 June 2022
Published: 21 June 2022
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viruses
Article
Grapevine Leafroll-Associated Virus 3 Genotype Influences
Foliar Symptom Development in New Zealand Vineyards
Kar Mun Chooi 1, * , Vaughn A. Bell 2, Arnaud G. Blouin 3, Daniel Cohen 1, Dion Mundy 4,
Warwick Henshall 1and Robin M. MacDiarmid 1,5
1The New Zealand Institute for Plant and Food Research Limited (PFR), Private Bag 92169,
Auckland 1142, New Zealand; dan.cohen@outlook.co.nz (D.C.); whenshall@actrix.co.nz (W.H.);
robin.macdiarmid@plantandfood.co.nz (R.M.M.)
2The New Zealand Institute for Plant and Food Research Limited (PFR), Private Bag 1401,
Havelock North 4157, New Zealand; vaughn.bell@plantandfood.co.nz
3Virology-Phytoplasmology Laboratory, Agroscope, 1260 Nyon, Switzerland;
arnaud.blouin@agroscope.admin.ch
4The New Zealand Institute for Plant and Food Research Limited (PFR), P.O. Box 845,
Blenheim 7240, New Zealand; dion.mundy@plantandfood.co.nz
5School of Biological Sciences, The University of Auckland, Private Bag 92019, Auckland 1142, New Zealand
*Correspondence: karmun.chooi@plantandfood.co.nz
Abstract:
Grapevine leafroll disease (GLD) constrains wine production worldwide. In New Zealand,
the main causal agent of GLD is grapevine leafroll-associated virus 3 (GLRaV-3). To control GLD,
an integrated management program is used and includes removing (roguing) GLRaV-3-infected
vines from the vineyard. The classical foliar symptoms from virus-infected red-berry cultivars are
leaves with dark red intervein, green veins, and downward rolling of margins. Growers use these
phenotypic cues to undertake visual symptom identification (VSI) for GLD. However, the influence
of the known large genetic variation among GLRaV-3 isolates on the foliar symptoms from different
grapevine cultivars remains undescribed, especially in cool-climate growing environments, such
as New Zealand. Over three vintages (2015, 2016, and 2017), VSI for GLD was undertaken at three
field sites in New Zealand (Auckland, Hawke’s Bay, and Marlborough), each including four cultivars
(Merlot, Pinot noir, Sauvignon blanc, and Pinot gris) infected with three GLRaV-3 genotypes (Groups
I, VI, and X) or GLRaV-3-uninfected control plants. Throughout this study, no visual symptoms
were observed on white-berry cultivars infected with GLRaV-3. For red-berry cultivars, the greatest
variability in observed foliar symptoms among regional study sites, cultivars, and GLRaV-3 genotypes
was observed early in the growing season. In particular, Group X had significantly delayed symptom
expression across all three sites compared with Groups I and VI. As the newly infected, young vines
matured in years 2 and 3, the GLRaV-3 genotype, cultivar, region, and environmental conditions had
minimal influence on the accuracy of VSI, with consistently high (>95%) within-vintage identification
by the end of each vintage. The results from this study strongly support the use of VSI for the GLD
management of red-berry cultivar grapevines, Merlot and Pinot noir, as a reliable and cost-effective
tool against GLD.
Keywords:
grapevine leafroll disease; grapevine leafroll-associated virus 3; visual symptoms
identification; host–virus interaction
1. Introduction
When plants become infected with a pathogen, they can display a range of symptoms,
such as deformation, discoloration, and wilting, or death of organ parts, whole organs, or
the entire plant. Symptom recognition and evaluation of its intensity is often employed in
plant virology. For example, visual cues are the basis of the biological indexing process used
to monitor regulated viruses in plant material for the trade or production of clean stock [
1
].
Viruses 2022,14, 1348. https://doi.org/10.3390/v14071348 https://www.mdpi.com/journal/viruses
Viruses 2022,14, 1348 2 of 19
Furthermore, visual symptom identification (VSI) is widely used by grape growers to
identify infected plants for removal [2,3].
Grapevine leafroll disease (GLD) is an economically significant viral disease that can
lead to reduced vine vigor and longevity and reduced fruit yield and quality, which in turn
negatively affects wine quality [
4
–
9
]. In New Zealand and worldwide, the leading causal
agent for this disease is the grapevine leafroll-associated virus 3 (GLRaV-3). GLRaV-3 is
the type member of the genus Ampelovirus, family Closteroviridae [
10
]. GLRaV-3 infection
in grapevines has been shown to affect various aspects of the crop and grapevine health.
A prominent visual GLRaV-3 foliar symptom is the eponymous downward rolling of
the leaf margins and for red cultivar grapevines, the premature reddening of the leaves
that starts as reddish spots that progressively spread to cover most, if not all, of the leaf
surface, with the primary veins remaining green [
5
,
11
,
12
]. The leaves of some white-
berry cultivars, such as Chardonnay, have been reported to turn yellow or chlorotic, but
they do not exhibit the leaf reddening described for red-berry cultivars [
5
]. GLRaV-3
symptom development in the leaves of red cultivar grapevines has an asymptomatic period
followed by a symptomatic phase (post-véraison) [
4
,
6
]. It is postulated that post-véraison,
the phloem-limited GLRaV-3 causes an imbalance in the carbohydrate source-to-sink
relationship, and this imbalance plays a key role in prematurely triggering the flavonoid
biosynthesis [
4
,
13
–
15
]. The mechanism by which GLRaV-3 triggers the sugar imbalance
has not been described.
This distinct foliar change in red cultivar grapevines is used as a cue for VSI of GLD in
the New Zealand leafroll management strategy [
2
] and is becoming a more common tool
in other countries for growers to identify virus infection for vine removal (roguing) [
16
].
VSI of red cultivars by a trained assessor has been demonstrated to be of high efficacy for
four different red cultivars, with 99.9% of the 114,782 grapevines in one study agreeing
with the laboratory enzyme-linked immunosorbent assay (ELISA) test [
2
]. It is important
that the assessors who perform VSI are be well-trained to ensure the viral symptoms are
not confused with abiotic stresses, such as nutrient deficiencies (e.g., magnesium and
potassium) and girdling of canes, which often initiate flavonoid biosynthesis and generate
similar reddening of leaves. GLRaV-3 has been reported to cause an upregulation of
flavonoid biosynthesis genes in symptomatic leaves from red cultivar grapevines compared
with the respective healthy leaves [
13
,
17
]. Conversely, the lack of the regulatory genes
MYBA1 and MYBA2 in white cultivar grapevines likely leads to the lack of red pigment
expression in their leaves [18–20].
To date, comparative studies regarding the impact of GLD on grapevines have pro-
duced conflicting results and differing interpretations [
4
,
5
,
12
,
21
]. Discrepancies are likely
because of the varying biotic influences (e.g., virus genotype, cultivar, duration of infection,
scion/rootstock, and combined infection with other viruses) and/or abiotic influences
(e.g., climatic conditions, soil mineral content, and vine management practices). These
complexities make it particularly difficult to gauge the possible impacts one virus has on
the grapevine and berry/wine quality when comparing studies.
Genetic variation in plant virus populations can lead to differences in symptom
development, as demonstrated by the well-studied citrus tristeza virus (CTV), another
phloem-limited virus in the family Closteroviridae. CTV genotypes can cause different
symptoms across different citrus species/cultivars, and different CTV genotypes can cause
different symptoms in the same citrus species/cultivars [
22
]. The GLRaV-3 population
worldwide shows substantially high genetic diversity, comprising at least eight different
phylogenetic groups (Groups I, II, III, V, VI, VII, IX, and X) that can differ by more than
20% across the entire genome [
23
]. GLRaV-3 genotypes representative of Groups I to III, V,
VI, and X have been identified in New Zealand commercial vineyards [
24
,
25
]. GLRaV-3
Groups I, VI, and X exhibit more than a 20% difference from each other based on the entire
genome. To date, little research has been conducted to understand the effects of this high
genetic diversity on symptom development [
24
,
26
–
34
]. However, results from a previous
study suggest that VSI-based vine roguing may be biased toward severe strains and that the
Viruses 2022,14, 1348 3 of 19
prominent distribution of GLRaV-3 Group X in the field may result from milder symptom
expression (missed by the assessor) and/or more efficient vector transmission [26].
This study aimed to understand the effects of three distinct GLRaV-3 genotypes
(Groups I, VI, and X) on the development of grapevine foliar symptoms and how they might
influence the accuracy of VSI for GLD. Subsequent research measuring grapevine growth
and berry composition from white- and red-berry cultivars in New Zealand conditions
has been undertaken and will be reported separately. Here, we present new insights into
the complex and intricate plant, virus, and environment interactions that underlie the
host response to GLD and the impacts that GLRaV-3 genotypes have on VSI outcomes in
New Zealand.
2. Materials and Methods
To investigate how three distinct GLRaV-3 genotypes influence foliar symptom devel-
opment, we established plantings of four grapevine cultivars (Vitis vinifera Merlot, Pinot
noir, Pinot gris, and Sauvignon blanc) on the same rootstock clone (3309 Couderc (3309C);
V. riparia
×
V. rupestris) in three New Zealand winegrowing regions (Auckland, Hawke’s
Bay, and Marlborough). These grapevines were purposefully and synchronously infected
with the same source material (single infections each of Groups I, VI, and X). In addition,
the effects of combinations of these three distinct GLRaV-3 genotypes (i.e., dual infections
of Groups I and VI, Groups I and X, and Groups VI and X) on the leaf symptomology
within infected grapevines were examined at the Auckland site, across all four cultivars.
2.1. Plant Material and Propagation
To establish the tri-regional trial sites, three key steps were completed: (i) selection
and propagation of appropriate GLRaV-3-infected material, (ii) green grafting inoculations
of selected GLRaV-3-infected material to four grapevine cultivars, and (iii) planting and
maintenance of each field trial plot.
A preliminary screen of prospective grapevines displaying typical GLD foliar symp-
toms that could be used as a source of virus inoculum from New Zealand commercial
vineyards was conducted by double antibody sandwich ELISA (DAS-ELISA) [
35
] and con-
ventional reverse transcription polymerase chain reaction (RT-PCR) using GLRaV-3-specific
primers as described previously [
26
]. Following this testing, cane material from selected
vines with GLRaV-3 infections of interest were collected, self-rooted, potted, and grown in
a glasshouse.
To confirm the virus population in the source plant material, the propagated glasshouse
plants were screened by DAS-ELISA [
35
], conventional and real-time RT-PCR assays using
GLRaV-3-specific primers [
26
,
27
], and high throughput sequencing (HTS) of the double-
stranded RNA following a previously described protocol [
36
,
37
]. The following set of rules
in regard to virus population, in order of priority, was used to select the most suitable
source of propagated plant material for the establishment of the regional sites. The selected
source material: (i) had to have the correct GLRaV-3 infection of interest and no other
grapevine leafroll virus or vitivirus, (ii) had to have the presence of grapevine rupestris
stem pitting-associated virus (GRSPaV) was accepted, and (iii) must not have grapevine
fleck virus. Refer to Table S1 for HTS results from source plant material screening and
plant section.
Green cane material from the selected glasshouse GLRaV-3-source grapevines (single-
and dual-infected) were green-grafted on top of the potted grapevines certified as GLRaV-3-
free (potted plants were sourced from and grafts performed by Riversun Nursery Limited,
Gisborne; Table S2 presents green graft success rates). The potted grapevines consisted of
four scion cultivars imported from Entav-INRA: Merlot 181, Pinot noir 777, Pinot gris 52,
and Sauvignon blanc 376. To generate dual GLRaV-3 Groups I and VI infected grapevines,
after 8 weeks, a second green bud from plant material positive for Group VI was grafted
on the 3309C rootstock of grapevines with a successful first graft with plant material
positive for Group I. Additionally, after the initial planting in 2014, we sought to resolve
Viruses 2022,14, 1348 4 of 19
a shortfall in grapevines, with additional green grafting performed and planted in 2015
(Table S3). For these additional graft events, the grapevines were tested by DAS-ELISA [
35
]
and subsequently by quantitative real-time RT-PCR (RT-qPCR) using generic and variant
specific primer sets [
26
,
27
], and high-resolution melting curve (HRM) analyses (Section 2.6)
were conducted prior to planting at the Auckland field plot.
2.2. Site Location and Study Configuration
A single trial site was established in each of the three wine grape regions in New
Zealand: Auckland (36
◦
49
0
22.03
00
S, 174
◦
28
0
35.13
00
E), Hawke’s Bay (39
◦
39
0
13.56
00
S,
176
◦
52
0
34.74
00
E), and Marlborough (41
◦
29
0
26.26
00
S, 173
◦
57
0
20.06
00
E). At each site, four
grapevine cultivars (V. vinifera Merlot, Pinot noir, Pinot gris, and Sauvignon blanc) infected
with single infections of the GLRaV-3 genotype representative of Groups I, VI, and X [
23
]
and associated healthy controls for each cultivar were planted. In addition, at the Auckland
site, V. vinifera Merlot, Pinot noir, Pinot gris, and Sauvignon blanc grapevines were infected
with two GLRaV-3 genotypes (Groups I and VI, Groups I and X, and Groups VI and X),
and their respective healthy controls were planted. Twenty biological replicates for each
virus treatment and cultivar at each site were prepared. However, grapevine mortality
after planting and failure to inoculate the GLRaV-3 genotype resulted in some treatments at
each site varying between 16 and 20 biological replicates, with the exception of Pinot gris
inoculated with Groups I and VI where only 13 plants were obtained (Tables S3 and S4).
To ensure the grapevines were spread evenly throughout each of the sites and to
control for possible terrain differences, a planting plan based on the Williams Design [
38
]
was implemented. Each bay (an area between two trellis posts along a vine row) contained
five vines of the same treatment (GLRaV-3-infected or healthy control) and a single healthy
grapevine (of the same cultivar) that acted as a “buffer grapevine”. The “buffer grapevines”
were regularly visually assessed, and ELISA tested to determine if there was secondary
mealybug (insect vectors of GLRaV-3) spread occurring within the field plot.
2.3. Planting and Maintenance of Each Field Trial Plot
In late January to early February of 2014, a total of 1,432 grapevines (i.e., grapevines
that showed successful green grafts with growth of grafted shoots (single and dual GLRaV-
3 infections), associated healthy grapevines, and “buffer grapevines”) were planted at the
three sites. Early in vintage 2015 (i.e., December 2014), an additional 107 grapevines were
planted across the three regional sites. These replaced grapevines that did not survive the
first planting or grapevines with additional bud grafts to fulfil the replicate numbers for the
dual GLRaV-3-infected grapevines. After the grapevines were planted, the grafted shoot
(i.e., the source of GLRaV-3 inoculum) was removed from the grapevines to ensure only
shoots from the intended scion grew.
Over the three years, grapevines at each field plot were maintained in accordance with
New Zealand wine industry standards. This involved standard drip irrigation, adopting
industry fungicide and insecticide spray regime recommendations, and standard viticul-
tural practices for 1-cane laid grapevines. To prepare grapevines for vintage 2015, all vines
were pruned to two buds (to encourage strong growth of a single shoot to be laid along
the fruiting wire). To prepare grapevines for vintages 2016 and 2017, grapevine pruning
options were as follows: grapevines that did not reach the fruiting wire or had poor cane
development (i.e., small cane diameter) were pruned to two buds; for grapevines that
reached the fruiting wire, a single cane was laid on the wire and pruned to eight buds.
To ensure continuity of the GLRaV-3 status among the virus-infected, healthy control,
and buffer vines, grapevine leaf or cane material was sampled annually and tested by
DAS-ELISA [35]. Selected samples were also tested by RT-qPCR [27].
2.4. Climate Data
Throughout this project, weather data were collected for the Hawke’s Bay and Marl-
borough sites from long-established weather stations that were either on-site (Hawke’s
Viruses 2022,14, 1348 5 of 19
Bay) or less than 2 km from the site (Marlborough). In Auckland, an on-site weather
station collected temperature, relative humidity, and rainfall data on an hourly basis from
18 November 2015. Data from the Auckland site were manually acquired from the weather
station; data from the Hawke’s Bay and Marlborough weather stations were acquired
through the MetWatch Online website (http://www.hortplus.metwatch.co.nz/index.php;
assessed on 2 September 2021). The hourly data were summarized on a monthly basis, and
general indices were calculated.
To gain an indication of annual climatic variation at each site and the extent of the
climatic difference among sites, the mean temperature of the warmest month (MTWM)
(
◦
C), mean temperature of the coldest month (MTCM) (
◦
C), and annual rainfall (mm)
were calculated. In addition, commonly used methods to assess site quality for growing
grapevines and the particular cultivar(s) to be grown at a site, such as growing degree
days (GDDs) (between 1 September and 30 April, 10
◦
C base temperature) and latitude
temperature index (LTI) [39], were assessed from available data.
2.5. Visual Symptom Identification (VSI)
Southern Hemisphere growing seasons (September to April) are hereafter referred
to as vintages for 2015, 2016, and 2017. During each of the three vintages reported, visual
assessments of foliar symptoms in red and white cultivar grapevines were performed by
the same VSI-trained researchers stationed at each field site [
2
]. The visual symptoms on
grapevines were monitored fortnightly to monthly, as required, from late December/early
January to mid-April. For red-berry grapevines, foliar symptoms were assessed based on a
symptom score range from 0 (no symptoms) to 3 (severe symptoms) (Figure 1a); the foliar
symptoms in white-berry grapevines were assessed based on a “yes” or “no” criterion. A
red-berry grapevine was visually identified as GLRaV-3-positive when vines were assessed
as a “2” or “3” based on the symptom assessment scale. The locations of observed foliar
symptoms were also recorded. In the 2015 vintage, the young canopy was divided into two
zones: (i) the base zone and (ii) the middle to top zone of the growing shoot (Figure 1b). At
the Auckland and Hawke’s Bay sites, as the vines matured in vintages 2016 and 2017, the
canopy was sectioned conceptually into six zones (three zones spanning the length of the
fruiting wire and the other three directly above, comprising the upper canopy) (Figure 1c).
2.6. Quantitative Real-Time RT-PCR (RT-qPCR) and High-Resolution Melting Curve
(HRM) Analysis
Approximately 100 mg of plant tissue was used for total RNA extractions, using a
modified cetyltrimethylammonium bromide (CTAB) extraction method [
40
]. In addition
to the RT-PCR and real-time RT-PCR assays using GLRaV-3-specific primers [
26
,
27
], a
high-resolution melting curve (HRM) analysis was performed to assist with confirming
the GLRaV-3 genotype infection status of single- and mixed-infected grapevines. Reverse
transcription was performed using the designed generic virus-specific reverse primer
(GLR3-GEN-9730R: 5
0
-CCTTCAGGACCTAGCACTTTCAGCG-3
0
) and the standard man-
ufacturer’s protocol (Invitrogen, Carlsbad, CA, USA). The PCR amplification and high-
resolution melting curve analysis were carried out in 10
µ
L reactions using the HOT FIREPol
®
EvaGreen
®
HRM Mix (Solis BioDyne, Tartu, Estonia) and performed in an Illumina Eco
™
Real-Time PCR System. Each reaction also contained 200–400 ng of total RNA extract
and 200 nM of each forward (GLR3-GEN-9558F: 5
0
-GTCTTTGGTGGACGACGGGAG-3
0
)
and reverse (GLR3-GEN-9730R) primer. Thermocycling conditions were 95
◦
C for 5 min,
40 cycles of 95
◦
C for 10 s, annealing at 60
◦
C for 20 s, and extension at 72
◦
C for 20 s, and
the final high-resolution melt curve analysis was 95
◦
C for 15 s, 55
◦
C for 15 s, and 95
◦
C
for 15 s.
Viruses 2022,14, 1348 6 of 19
Figure 1.
Grapevine leafroll disease (GLD) visual symptom identification (VSI) assessment criteria for
all sites during vintages 2015, 2016, and 2017. (
a
) Symptom scale (0 to 3) for GLD foliar symptoms on
red-berry grapevine leaves. The images were used as visual comparisons to enable standardization
of VSI scoring at all three regional sites. (
b
) The young grapevine canopy of red-berry cultivars
(assessed in the 2015 vintage) was divided into two zones: base (B) and middle to top (M + T) canopy
zones, as indicated by the white dotted boxes. (
c
) In vintages 2016 and 2017, the mature grapevine
canopy of red-berry cultivars was divided into six zones for VSI, as indicated by the white dotted
boxes. All photographs are of Vitis vinifera Merlot.
3. Results
3.1. Site Climate Based on Air Temperature and Rainfall
Over the data collection time, at each site, the mean monthly temperatures (MTWMs
and MTCMs) were similar among vintages (Table 1). The MTWM was similar across the
three sites, while the Auckland site had a higher MTCM than the other two sites. The
annual rainfall increased gradually over the 3-year study for all three sites. Auckland had
the highest annual rainfall among the three sites, with at least 182% more rainfall than
Hawke’s Bay and Marlborough (ranged between 182 and 259%). The Hawke’s Bay site
generally received more rainfall than the Marlborough site (112–159% more). The GDDs
were generally similar across the three sites, with the exception of the 2017 vintage where
the Hawke’s Bay site had significantly higher GDDs than the other two sites. Consis-
tently, Auckland and Hawke’s Bay had a higher LTI than the Marlborough site across all
three vintages.
Viruses 2022,14, 1348 7 of 19
Table 1.
Climate data from the three grape-growing sites collected in vintages 2015, 2016, and 2017,
where possible. Mean temperature of the warmest month (MTWM) (
◦
C), Mean temperature of the
coldest month (MTCM) (
◦
C), Annual rainfall (mm), Growing Degree Days (GDDs) (
◦
C), and Latitude
temperature index (LTI).
Auckland Hawke’s Bay Marlborough
2015 vintage
MTWM (◦C) n/a 18.4 18.8
MTCM (◦C) n/a 7.9 7.4
Annual rainfall (mm) n/a 607.8 381.6
GDDs n/a 1357.3 1367.2
LTI n/a 374.4 347.8
2016 vintage
MTWM (◦C) 19.9 20.1 20.0
MTCM (◦C) 10.3 8.8 8.3
Annual rainfall (mm) 1276 661.9 590.8
GDDs (from 18 November 2015) 1277.8 1183.6 1167.6
GDDs (complete vintage) n/a 1357.3 1386.5
LTI 461.1 409.0 370
2017 vintage
MTWM (◦C) 18.2 19.7 18.6
MTCM (◦C) 10.2 8.0 7.9
Annual rainfall (mm) 1538 #846.6 591.4
GDDs 1405.6 1614.1 1368
LTI 431.0 400.9 344.1
n/a, not applicable as data were not available because the weather station at the Auckland field site was only
installed on 18 November 2015.
#
Missing rainfall data from the weather station at the Auckland field trial site
were supplemented with data from weather station located in Mt Albert, Auckland (~24 km away).
3.2. Visual Symptom Identification (VSI) of Grapevines for Grapevine Leafroll Disease (GLD)
Over the three years of VSI for GLD foliar symptoms at the three regional sites, GLD
was observed only among the red cultivars, Merlot and Pinot noir grapevines, known to
be infected with GLRaV-3 (single or dual GLRaV-3 genotype-infected). No specific foliar
symptoms were observed among (a) the white cultivars (Pinot gris and Sauvignon blanc)
infected with GLRaV-3 (single or dual GLRaV-3-infected), (b) among the GLRaV-3-negative
(virus-free) red cultivar grapevines, or (c) among the buffer grapevines. The absence of
GLRaV-3 in the buffer and the nongraft-inoculated vines was confirmed by DAS-ELISA
and RT-qPCR. Thus, there was no evidence of a vector-mediated spread of GLRaV-3 at any
regional site.
3.2.1. Visual Symptom Identification of Grapevines Infected with One GLRaV-3 Genotype
Over the three vintages at all sites, positive VSI, and thereby the VSI accuracy, in-
creased gradually over each vintage (Figure 2). The lowest proportions of positively
identified GLRaV-3-infected red cultivar grapevines (i.e., lowest VSI accuracy) were at
early inspection time points, and the highest and most consistent identifications of GLRaV-
3-infected grapevines (i.e., highest and most consistent VSI accuracy) were late in each
vintage (mid-March–April, from Julian date 70 onward) (Figure 2).
Viruses 2022,14, 1348 8 of 19
Figure 2.
Visual symptom identification of grapevine leafroll-associated virus 3 (GLRaV-3) in the field
trial across three regional locations (Auckland, Hawke’s Bay, and Marlborough) and two red-berry
cultivars (Merlot and Pinot noir) for vintage 2015 (triangle points), 2016 (square points), and 2017
(circle points). Data are percentages of Merlot and Pinot noir vines (left and right, respectively) singly
infected GLRaV-3 (irrespective of genotype) that were visually identified as GLRaV-3-positive based
on distinctive foliar changes (i.e., assessed as a “2” or “3” based on the symptom assessment scale).
Over the three vintages, no visual symptoms were observed on any white-berry cultivars (Pinot gris
and Sauvignon blanc) at any of the regional locations; therefore the results are not displayed.
At all sites, a noticeably higher proportion of red cultivar grapevines showed foliar
symptoms earlier in vintage 2017 (year 3 of visual assessments) than in assessments under-
taken in the 2015 vintage (year 1 of visual assessments) and 2016 vintage (year 2 of visual
assessments). In addition, GLD symptoms were generally observed earlier in each vin-
tage among the Merlot grapevines infected with GLRaV-3 than among infected Pinot noir
vines (Figure 2).
In each vintage, regional differences in the proportion of GLRaV-3-infected Merlot
and Pinot noir grapevines positively identified with GLD foliar symptoms were observed
during early VSI time points (Figure 2). For example, at the first assessment in February of
the 2016 vintage (Julian days 35, 36, and 41 for the Marlborough, Hawke’s Bay, and Auck-
land assessments, respectively), only 1 out of 59 Merlot GLRaV-3-infected grapevines
were observed to have GLD symptoms at the Marlborough site compared with 40%
(24 out of 60) and 98% (57 out of 58) at the Auckland and Hawke’s Bay sites, respec-
tively. This difference could not be fully explained by variable VSI assessor capability.
During the 2016 vintage (Julian day 48), both the Auckland and Hawke’s Bay assessors
inspected the grapevines at the Auckland site independently of one another. The Hawke’s
Bay assessor positively identified 68% of the Merlot (41 out of 60) and 42% of the Pinot
noir (24 out of 57) GLRaV-3-infected grapevines; the Auckland assessor visually diagnosed
Viruses 2022,14, 1348 9 of 19
67% (40 out of 60) and 35% (20 out of 57) of the Merlot and Pinot noir GLRaV-3-infected
grapevines, respectively.
3.2.2. GLD Symptoms Based on Virus Genotype and Spread of Symptoms over the Canopy
At all three regional study sites, the foliar symptoms for both Merlot and Pinot noir cul-
tivar grapevines infected with the Group I or VI genotypes expressed symptoms earlier in
the vintage than Group X-infected grapevines (Figure 3displays results from the Hawke’s
Bay site; results from Auckland and Marlborough are presented in Figures S1 and S2). For
all GLRaV-3 genotypes, a similar trend for GLD vertical progression of symptom expression
across the canopy was observed during vintages 2016 and 2017. Initially, symptoms were
observed at the basal portions of the canopy (canopy region nearest the laid cane, referred
to as Sections 1, 3, and 5 in this study), but as each growing season advanced, viral symp-
toms were progressively observed in the upper canopy (Sections 2, 4, and 6), with overall
symptom expression more consistent throughout the canopy late in the vintage (Figure 4,
Merlot grapevines at the Hawke’s Bay site; Figure S3, Pinot noir grapevines at the Hawke’s
Bay site; and Figures S4 and S5, Merlot and Pinot noir grapevines at the Auckland site).
As with the delayed assessment of symptomatic Group X-infected grapevines, the spread
of visible foliar symptoms from the basal to the upper sections of the grapevine canopy
was also slower in Group X-infected Merlot and Pinot noir grapevines relative to the other
virus genotypes in this study. No apparent difference in the visible foliar symptoms ex-
pressed by the different GLRaV-3 genotypes horizontally across the canopies was observed
(Figures S6 and S7). A similar proportion of Merlot and Pinot noir grapevines showed
GLD symptoms in shoots closest to the trunk compared with shoots furthest away from
the trunk at each time point over each growing season (Figures S6 and S7).
Figure 3.
Visual identification of grapevine leafroll-associated virus 3 (GLRaV-3) genotypes at
the Hawke’s Bay site for the red-berry cultivars Merlot (left panel) and Pinot noir (right panel) in
Viruses 2022,14, 1348 10 of 19
vintages 2015, 2016, and 2017. Generally, in all vintages, delayed symptom expression was ob-
served for Merlot and Pinot noir (especially in vintages 2016, 2017) infected with Group X (diamond
points) relative to Group I (star points) and Group VI (triangle points) virus infections. This re-
duced the overall percentage of GLRaV-3-infected vines positively identified by foliar symptoms,
with the exception of vintage 2015, where the percentage of Group X-infected Pinot noir vines pos-
itively identified for disease based on foliar symptoms was greater than that for Group VI in the
same cultivar.
Figure 4.
Foliar symptoms of the grapevine leafroll-associated virus 3 (GLRaV-3) Group X genotype
appeared later than the Group I and IV genotypes symptoms from infected Merlot grapevines, as
recorded in the Hawke’s Bay study site during vintages 2016 (left panel) and 2017 (right panel). For
all genotypes, the expression of foliar symptoms moved from the laid cordon (base of the canopy)
upward, as the growing season progressed. Presented are the cumulative percentages of grapevines
infected with genotypes Group I (star points), Group VI (triangle points), and Group X (diamond
points) with observable leafroll symptoms in all basal canopy sectors (demarcated as Sections 1, 3,
and 5), the first symptom appearance in the upper canopy sectors (demarcated as Sections 2, 4 and 6),
and symptoms in all canopy sectors (demarcated Sections 1 to 6).
3.3. Visual Symptom Identification of Grapevines Infected with Two GLRaV-3 Genotypes
Similar to the grapevines infected with a single GLRaV-3 genotype, the VSI accuracy
of dual GLRaV-3-infected grapevines increased gradually over each vintage, and late
in each vintage the most consistent detection of GLD foliar symptoms was observed
(Figure S8). This gradual increase in the proportion of positively identified Merlot and
Pinot noir grapevines infected with two GLRaV-3 genotypes matched those of grapevines
infected with one GLRaV-3 genotype in vintages 2016 and 2017 (years 2 and 3) (Figure S8).
The exception was year 1 (vintage 2015), where a lower proportion of Merlot and Pinot noir
vines with dual GLRaV-3 infections showed foliar symptoms compared with grapevines
Viruses 2022,14, 1348 11 of 19
containing a single GLRaV-3 genotype, likely because some of the double-inoculated vines
were produced later.
Differences between grapevines infected with one GLRaV-3 genotype and their coun-
terpart grapevines infected with the same genotype in a coinfection with another
GLRaV-3 genotype were often observed at early VSI time points in each vintage
(Figures 5, S9 and S10). Notably, lower proportions of Merlot grapevines with a coin-
fection of the Group I and X genotypes were consistently observed with leafroll symp-
toms at the early growing season assessment time points (between Julian days 30 and 60)
compared with proportions of Merlot grapevines infected singularly with Group I or coin-
fected with Groups I and VI) (Figure S9). Moreover, generally fewer red cultivar grapevines
with Group VI coinfected with Groups I or X were observed with leafroll symptoms com-
pared with grapevines infected with Group VI only. In particular, the visual symptom
expression of GLRaV-3 in Merlot and Pinot noir grapevines was less pronounced when
Group VI was coinfected with Group X in the 2017 vintage (Figure S10). For example, at
Julian day 38, 44% Merlot and 45% Pinot noir grapevines with dual Group VI and X infec-
tion showed GLD symptoms, compared with 70% Merlot and 82% Pinot noir grapevines
infected with only the GLRaV-3 Group VI genotype. Conversely, for two consecutive
years (vintages 2016 and 2017), at early vintage time points, a greater proportion of Merlot
and Pinot noir grapevines coinfected with Group X and either Group I or Group VI was
observed with leafroll symptoms compared with the respective grapevines infected with
Group X only (Figure 5).
Figure 5.
Visual identification of grapevine leafroll-associated virus 3 (GLRaV-3) from Merlot (left
panel) and Pinot noir (right panel) grapevines infected with only the GLRaV-3 Group X genotype
(diamond points) and with Group X in a dual infection with Group I (“X” points) or Group VI
(“ ” points) genotype at the Auckland site, for the vintages 2015, 2016, and 2017.
Viruses 2022,14, 1348 12 of 19
4. Discussion
Studying the disease etiology of phloem-limited viruses, such as GLRaV-3, that infect
perennial plants is difficult in the natural environment without access to an infectious viral
clone that infects the natural host systemically. In most GLRaV-3 studies, important factors,
such as virus genotype(s), time of infection, duration of infection, geographic location,
presence of other viruses, and/or scion/rootstock, are not considered [
4
,
5
,
12
,
21
]. This
lack of comparative rigor has probably led to discrepant findings between studies. To
our knowledge, this study is the first field trial that used purposefully infected different
grapevine cultivars with different GLRaV-3 genotypes by grafting and planting grapevines
in three geographically distinct regions and that assessed disease expression over multiple
vintages. Here, we discuss critically how the physiological data presented in this study
reflect existing commentaries and hypotheses for GLD foliar symptom development; we
describe new insights into the importance of plant–virus interactions for disease expression
and discuss the practical implications of virus genotypes on the utility of VSI for GLD
management in the vineyard.
4.1. Véraison Coincides with GLD Foliar Symptoms in Red-Berry Grapevines
This study supports the current GLRaV-3 hypothesis that GLD foliar symptoms
of red cultivar grapevines have two phases: a pre-véraison asymptomatic phase and
a postvéraison symptomatic phase [
4
,
6
]. After 3 years of observations, typical leafroll
foliar symptoms in Merlot and Pinot noir red-berry cultivars were only apparent post-
véraison (after mid-January in New Zealand) at all three trial sites (Figure 2). There were
no asymptomatic vines from vines known to be infected with GLRaV-3. Even after 8 years
from the initial infection, no foliar symptoms were observed prior to véraison in the 2021
vintage (K. Chooi personal observation; data not shown).
In vintages 2016 and 2017, in particular, a higher proportion of grapevines was ob-
served with foliar symptoms earlier in the season, which supports previous observations
that older infections of GLRaV-3 are more likely to appear earlier in the growing season [
2
].
Based on our results and current knowledge, there appears to be a strong temporal link
between GLD foliar symptoms caused by GLRaV-3 and carbohydrate movement within
the grapevines in New Zealand. At véraison, berries undergo a rapid size increase accom-
panied by turgor loss, softening, sugar accumulation, and color accumulation in red-berry
cultivars, and xylem hydraulic conductance decreases [41,42]. At the same time, the main
water transport pathway changes from xylem to phloem [
43
–
45
], and sugar transport shifts
from the symplastic to apoplastic pathway [
46
]. Is it possible the phloem-limited virus
influences this complex network of interactions and signals linked to véraison?
Notably, after 3 years of observations in New Zealand, GLRaV-3-infected Pinot gris
and Sauvignon blanc white-berry cultivars lacked any foliar symptoms, such as chlorosis
and leaf rolling. This lack of foliar symptoms contradicts other observations described
overseas, mostly on Chardonnay [
5
] and may in part be caused by additional factors, such
as cultivar/clone, environment, and the role of mixed viral infections. Although no foliar
symptoms were observed in these white cultivars in this study, more research is required
to understand whether GLRaV-3 influences other grapevine aspects. Subsequent research
measuring grapevine growth and berry composition from white- and red-berry cultivars in
New Zealand conditions has been undertaken and will be reported separately.
4.2. GLRaV-3 Genotypes, Foliar Symptom Development, and Potential Plant–Virus Interactions
Collected climate data provide some insights into the intra- and inter-site variability
in this study. Based on the grape-ripening capacity predictions using GDDs, all three
sites had similar ‘degree days’ or ‘heat units’ and therefore similar potential vine growth
and development (Table 1). With an LTI of less than 380, the Marlborough site could be
considered a warm climate region, where key cultivars, such as Merlot and Sauvignon
blanc, are often grown [
39
]. In contrast, the Auckland and Hawke’s Bay sites would be
considered as warm to hot climate regions (LTI greater than 380). Additionally, the three
Viruses 2022,14, 1348 13 of 19
field sites had notable differences in rainfall. Climatic and environmental aspects, such as
frost potential, wind, site elevation, and soil type, were not assessed.
Regardless of the environmental conditions measured at the three regional sites, all
red-berry grapevines showed foliar symptoms. Foliar symptom differences observed
among sites were negligible and far less important than those measured among treatments
(genotypes), which confirms in this case, that the genotype overcomes the environmental
factors. Foliar symptoms were consistently observed earlier in Group I-infected Merlot
and Pinot noir grapevines than in Group X-infected grapevines throughout this study
(Figures 3, S1 and S2). The difference in symptom expression among genotypes was most
prominent early in the vintage at the lower canopy sections and at the upper portions of the
canopy later in the vintage (Figures 4and S3–S5). This consistent observation of differential
onset of leaf reddening in grapevines infected with different GLRaV-3 genotypes implies
that GLRaV-3 and its genotype directly influence the timing and apparent severity of foliar
symptoms. The expression of leaf reddening in GLRaV-3-infected grapevines is assumed
to be caused by anthocyanin synthesis [
13
,
17
]; therefore, it is reasonable to assume that the
different timing of the onset of leaf reddening observed may be caused by an alteration in
the anthocyanin pathway.
Plant viruses are known to interact and influence an array of plant miRNAs, plant
pathways, and plant proteins to cause observable disease symptoms [
47
–
50
]. Phloem-
limited virus CTV and sugarcane yellow leaf virus have shown that they directly modulate
both sugar/carbohydrate and ROS synthesis and movements in plants [
49
,
51
,
52
], while
potyvirus and a bromoviridae virus have been demonstrated to modulate anthocyanin
biosynthesis through direct interaction of viral suppressors of RNA silencing (VSRs) en-
coded by these viruses with the anthocyanin regulatory pathway [
47
,
48
]. Higher starch and
sugar contents [
13
–
15
] and upregulation of genes associated with anthocyanin biosynthe-
sis [13,17,53] in symptomatic leaves from GLRaV-3-infected red-berry cultivars compared
with leaves of a similar age from healthy grapevines have been reported. Moreover, com-
parative studies of a known GLRaV-3 VSR protein, p19.7, encoded by different genotypes
using in vitro laboratory assays demonstrated variable VSR activity [28,54].
Rapid cell-to-cell and systemic virus movement may result in earlier and more ap-
parent symptom expression as shown for a modified tomato bushy stunt virus (TBSV)
coat protein (CP) [
55
,
56
]. Virion-associated proteins considered to play a role in virus
movement encoded by GLRaV-3 are highly divergent among the three genotypes used in
this study (between 5 and 22% amino acid differences). Differences in the N-terminus of
the GLRaV-3 CP likely alter the exposed structure(s) on the virion surface [
24
,
35
]. GLRaV-3
genotypes have been previously reported to be unevenly distributed and at different virus
titers in plants, particularly the Group X genotype that was inconsistently detected and at a
lower titer using molecular assays along a grapevine shoot compared with the detection of
Groups I and VI [
27
,
57
]. Collectively, the reported uneven GLRaV-3 genotype distribution
and variable GLRaV-3 VSR strengths support the hypothesis presented in the current
study that GLRaV-3 proteins influence host proteins and pathways with varying potencies,
which lead to different foliar symptoms through the season. Further studies using yeast
two-hybrid and bimolecular fluorescence complementation systems may elucidate the
specific host–virus protein interactions. Use of host proteins from different cultivars will
provide additional insights into variable cultivar responses to virus. In this study, the
Merlot GLRaV-3-infected grapevines developed foliar symptoms faster than Pinot noir
GLRaV-3-infected grapevines (Figure 2). Targeted in-vitro experiments, such as the over-
expression of VSR proteins in known plant systems where influences on the anthocyanin
biosynthesis can be measured, could also help elucidate the direct role of GLRaV-3 VSRs.
4.3. Dual Infections of Different GLRaV-3 Genotypes Influenced the Onset of GLD Foliar Symptom
Expression Compared with Single Infections of the Same GLRaV-3 Genotype
Generally, early in the vintage, the grapevines co-infected by Group X along with
Groups I or VI produced an intermediate response compared to the symptoms observed
Viruses 2022,14, 1348 14 of 19
from a single Group X GLRaV-3 infection and those from Group I or VI single infections
(Figures 5, S9 and S10). The observed variable symptom expression that was dependent on
the type of GLRaV-3 genotype(s) further supports the hypothesis that GLRaV-3 specifically
interacts with and influences plant host pathways and proteins. In particular, Groups I
and VI caused greater detrimental effects to the plant foliage than Group X. This result also
raises the questions: Do the Group I and VI genotypes have greater fitness with intra-plant
movement and plant defense suppression? Are Group I and VI genotypes able to occupy
and initiate plant stress responses in more plant cells, resulting in faster leaf reddening? Are
there synergistic/complementary virus–virus interactions among the GLRaV-3 genotypes
that aid the accumulation, movement, and/or virulence of the Group X genotype?
4.4. Visual Symptom Identification for GLD Foliar Symptoms in Red-Berry Grapevines Is an
Effective Method for GLD Management
VSI by trained staff for GLD foliar symptoms in red-berry grapevines has been demon-
strated to be an effective GLD management strategy to cost-effectively identify diseased
grapevines for removal and planting replacement grapevines [
2
]. Since the GLRaV-3
genotype was not taken into account by Bell et al. [
2
], the question remained whether the
GLRaV-3 genotype affects the expression and timing of symptom development and in
turn the efficacy of VSI. Our results, which include data from the two main grape-growing
regions of New Zealand (Hawke’s Bay and Marlborough) strongly support the use of VSI
by trained assessors to identify GLD in red-berry cultivars. This result was independent
of the GLRaV-3 genotype tested. Under our conditions, Group X-infected grapevines
expressed foliar symptoms, which suggest that the previously reported prominent distri-
bution of the Group X in a commercial vineyard [
26
] is more likely because of different
transmissibility rates by mealybugs rather than mild symptom expression influencing VSI.
We also confirmed the absence of any foliar symptoms among grapevines in either of the
two white cultivars used in this study. These observations across multiple years reiterated
the need for serological or molecular testing to identify virus-infected grapevines reliably.
We note, however, the additional cost of this testing may be a barrier to many growers
assessing virus in white cultivars, which as a virus reservoir has potential to threaten other
vine plantings in the vineyard.
For at least three dissimilar genotypes that represent the genetic extremes known to
occur in New Zealand, there is unlikely to be a GLRaV-3 genotype impact on the overall
detection of GLD in infected Merlot and Pinot noir grapevines if the VSI is performed as
recommended [
2
,
58
]. Visual assessments undertaken later in a vintage (before leaf fall) offer
the greatest prospect to identify a high percentage of symptomatic grapevines, particularly
newly infected grapevines, grapevines that have been leaf plucked (removing leaves from
the base of the canopy most likely to exhibit GLD symptoms), and/or grapevines infected
with genotypes that express late foliar symptoms, i.e., the Group X genotype. In the future,
there will need to be a better understanding of the compounding effects such as coinfection
with other grapevine viruses. An example is a dual GLRaV-1 infection that may have fewer
foliar symptoms that are less distinctive than GLRaV-3, thereby potentially reducing the
efficacy of VSI assessments.
5. Conclusions
In the absence of an infectious GLRaV-3 clone that can systemically infect grapevines,
this study demonstrated that GLRaV-3 causes the distinctive foliar changes in red-berry
cultivar grapevines. This is the first study to describe foliar symptoms monitored across
the canopy over a season and over multiple vintages. Regardless of the environmental
conditions, the foliar symptoms caused by the GLRaV-3 genotype were indistinguishable
from one another apart from the onset and general appearance of GLD symptoms across
the canopy. Foliar symptoms from Group I-infected Merlot and Pinot noir vines were
observed consistently earlier than Group X-infected grapevines. There are indications
the virus genotypes can influence the timing and severity of symptom expression when
Viruses 2022,14, 1348 15 of 19
coinfected in the same grapevine. GLRaV-3 did not cause distinctive foliar changes in the
white-berry cultivars included in this study. It will be of particular interest to understand
whether the different GLD foliar symptoms caused by the distinct genotypes in red-berry
cultivar grapevines or lack of foliar symptoms in white-berry cultivar grapevines are also
reflected in other aspects, such as plant physiology, including growth and berry yield and
quality, and in subsequent wine quality.
Supplementary Materials:
The following supporting information can be downloaded at: https://
www.mdpi.com/article/10.3390/v14071348/s1, Tables S1–S4, Figures S1–S10. Table S1: Detection of
grapevine leafroll-associated virus 3 and other grapevine viruses and/or viroids by high throughput
sequencing (HTS) in plant material from source plants used. Table S2: The success rate of the green-
graft inoculations for each of the grapevine leafroll-associated virus 3 (GLRaV-3) infection types
and grapevine cultivars. Table S3: Total number of biological replicates for each dual grapevine
leafroll-associated virus 3 genotype infection treatment and cultivar planted at the Auckland field
trial site. Grapevines were planted at the Auckland location across two vintages, 2014 and 2015.
Table S4: Total number of biological replicates (Rep.), number of vines tested negative for virus after
two consecutive years of laboratory testing (Neg.), and number of missing vines (Missing) for each
treatment and cultivar in the Auckland, Hawke’s Bay, and Marlborough grapevine leafroll-associated
virus 3 (GLRaV-3) field trial sites. Figure S1: Visual identification of grapevine leafroll associated
virus 3 (GLRaV-3) genotypes at the Auckland site for the red-berry cultivars Merlot (left panel) and
Pinot noir (right panel), in vintages 2015, 2016, and 2017. Generally, in all vintages, delayed symptom
expression was observed for Merlot and Pinot noir (especially in vintages 2016, 2017) infected with
Group X (diamond points) relative to Group I (star points) and Group VI (triangle points) virus
infections. This reduced the overall percentage of GLRaV-3-infected vines positively identified by
foliar symptoms. Figure S2: Visual identification of grapevine leafroll associated virus 3 (GLRaV-3)
genotypes at the Marlborough site for the red-berry cultivars Merlot (left panel) and Pinot noir (right
panel), in vintages 2015, 2016, and 2017. Generally, in all vintages, delayed symptom expression
was observed for Merlot and Pinot noir (especially in vintages 2016, 2017) infected with Group X
(diamond points) relative to Group I (star points) and Group VI (triangle points) virus infections. This
reduced the overall percentage of GLRaV-3-infected vines positively identified by foliar symptoms,
with the exception of vintage 2016, where the percentage of Group X-infected Merlot vines positively
identified for disease based on foliar symptoms was greater than that for Group I in the same cultivar
at the Julian day 53 (represents one extra vine identified). Figure S3: Foliar symptoms of the grapevine
leafroll-associated virus 3 (GLRaV-3) Group X genotype generally appeared later than the Groups
I and IV genotype symptoms from infected Pinot noir grapevines, as recorded in the Hawke’s Bay
study site during vintages 2016 (left panel) and 2017 (right panel). For all genotypes, the expression of
foliar symptoms moved from the laid cordon (base of the canopy) upwards, as the season progressed.
Presented are the cumulative percentages of grapevines infected with genotypes Group I (star points),
Group VI (triangle points), and Group X (diamond points) with observable leafroll symptoms in all
basal canopy sectors (demarcated as Sections 1, 3, and 5), the first symptom appearance in the upper
canopy sectors (demarcated as Sections 2, 4, and 6), and symptoms in all canopy sectors (demarcated
Sections 1 to 6). Figure S4: Foliar symptoms of the grapevine leafroll-associated virus 3 (GLRaV-3)
Group X genotype appeared later than the Groups I and IV genotype symptoms from infected Merlot
grapevines, as recorded in the Auckland study site during vintages 2016 (left panel) and 2017 (right
panel). For all genotypes, the expression of foliar symptoms moved from the laid cordon (base
of the canopy) upwards, as the season progressed. Presented are the cumulative percentages of
grapevines infected with genotypes Group I (star points), Group VI (triangle points), and Group
X (diamond points) with observable leafroll symptoms in all basal canopy sectors (demarcated as
Sections 1, 3, and 5), the first symptom appearance in the upper canopy sectors (demarcated as
Sections 2, 4, and 6), and symptoms in all canopy sectors (demarcated Sections 1 to 6). Figure S5:
Foliar symptoms of the grapevine leafroll-associated virus 3 (GLRaV-3) Group X genotype appeared
later than the Groups I and IV genotype symptoms from infected Pinot noir grapevines, as recorded
in the Auckland study site during vintage 2017. For all genotypes, the expression of foliar symptoms
moved from the laid cordon (base of the canopy) upwards, as the season progressed. Presented
are the cumulative percentages of grapevines infected with genotypes Group I (star points), Group
VI (triangle points), and Group X (diamond points) with observable leafroll symptoms in all basal
Viruses 2022,14, 1348 16 of 19
canopy sectors (demarcated as Sections 1, 3, and 5), the first symptom appearance in the upper
canopy sectors (demarcated as Sections 2, 4, and 6), and symptoms in all canopy sectors (demarcated
Sections 1 to 6). Figure S6: Visual identification of foliar symptoms from three canopy sections (V1,
V2, V3) of Merlot (left panel) and Pinot noir (right panel) grapevines infected with either the GLRaV-3
Group I (star points), Group VI (triangle points), and Group X (diamond points) genotype, for the
2016 vintage visual assessments at the Hawke’s Bay site. No apparent difference in the observable
foliar symptoms (either the first appearance of symptoms or full canopy coverage of symptoms)
among the three vertical canopy sections was observed. Similar cumulative percentage of grapevines
with observable symptoms from shoots closest to the trunk (V1) compared with shoots furthest
away from the trunk (V3) throughout the growing season. Figure S7: Visual identification of foliar
symptoms from three canopy sections (V1, V2, V3) of Merlot (left panel) and Pinot noir (right panel)
grapevines infected with either the GLRaV-3 Group I (star points), Group VI (triangle points), and
Group X (diamond points) genotype, for the 2017 vintage visual assessments at the Hawke’s Bay site.
No apparent difference in the observable foliar symptoms (either the first appearance of symptoms
or full canopy coverage of symptoms) among the three vertical canopy sections was observed. A
similar cumulative percentage of grapevines with observable symptoms from shoots closest to the
trunk (V1) compared with shoots furthest away from the trunk (V3) throughout the growing season.
Figure S8: Visual symptom identification of grapevine leafroll associated virus 3 (GLRaV-3) from
Merlot (left panel) and Pinot noir (right panel) grapevines infected with a single GLRaV-3 genotype
(square points) or with two GLRaV-3 genotypes (circle points) at the Auckland site, for the 2015,
2016, 2017 vintages. Figure S9: Visual symptom identification of grapevine leafroll associated virus 3
(GLRaV-3) from Merlot (left panel) and Pinot noir (right panel) grapevines infected with only the
GLRaV-3 Group I genotype (star points) and with Group I in a dual infection with Group VI (circle
points filled with a “+”) or Group X (“X” points) genotypes at the Auckland site, for the 2016 and
2017 vintages. Figure S10: Visual symptom identification of grapevine leafroll associated virus 3
(GLRaV-3) from Merlot (left panel) and Pinot noir (right panel) grapevines infected with only the
GLRaV-3 Group VI genotype (triangle points), and with Group VI in a dual infection with Group I
(circle points filled with a “+”) or Group X (“ ” points) genotypes at the Auckland site, for the 2016
and 2017 vintages.
Author Contributions:
Conceptualization, K.M.C., A.G.B., D.C., V.A.B. and R.M.M.; methodology,
K.M.C., A.G.B., D.C., D.M., W.H., V.A.B. and R.M.M.; formal analysis, K.M.C.; investigation, K.M.C.,
A.G.B., D.C., D.M., W.H. and V.A.B.; resources, K.M.C. and R.M.M.; data curation, K.M.C. and A.G.B.;
writing—original draft preparation, K.M.C., A.G.B., V.A.B. and R.M.M.; writing—review and editing,
K.M.C., A.G.B., D.C., D.M., W.H., V.A.B. and R.M.M.; visualization, K.M.C., A.G.B., V.A.B. and
R.M.M.; project administration, K.M.C. and R.M.M.; funding acquisition, K.M.C. and R.M.M. All
authors have read and agreed to the published version of the manuscript.
Funding:
K.M.C. was supported by postdoctoral fellowships from New Zealand Winegrowers and
the Agricultural and Marketing Research and Development Trust (AGMARDT). The resources were
funded by AGMARDT and Ministry of Business, Innovation and Employment (MBIE)—Strategic
Science Investment Funding.
Acknowledgments:
We thank Riversun Nurseries Limited for the assistance with grafting all the
grapevines for this study. We are grateful for the continued maintenance of the field sites by the
vineyard managers, especially Stephen Nobilo, Peter Welsh, and Tara Taylor. We also thank fellow
Plant & Food Research staff for assisting with the planting of grapevines and colleagues Mark Wohlers
for the advice with field site plot design, and Erik Rikkerink and Junqi Zhu for their helpful insights
and review of the manuscript prior to submission. We are grateful for the useful comments provided
by three anonymous reviewers.
Conflicts of Interest: The authors declare no conflict of interest.
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