Identification of powdery mildew resistance in wild grapevine (Vitis vinifera subsp. sylvestris Gmel Hegi) from Croatia and Bosnia and Herzegovina

Abstract

Wild grapevine ( Vitis vinifera subsp. sylvestris ) is widely recognized as an important source of resistance or tolerance genes for diseases and environmental stresses. Recent studies revealed partial resistance to powdery mildew ( Erysiphe necator, PM) in V. sylvestris from Central Asia. Here, we report resistance to PM of V. sylvestris collected from different regions of Croatia and in seedling populations established from in situ V. sylvestris accessions. Ninety-one in situ individuals and 67 V. sylvestris seedlings were evaluated for PM resistance according to OIV 455 descriptor. Three SSR markers (SC47-18, SC8-071-0014, and UDV-124) linked to PM resistance locus Ren1 were used to decipher allelic structure. Nine seedlings showed resistance in in vivo evaluations while leaf disk assays revealed three PM-resistant accessions. One V. vinifera cultivar used as a control for PM evaluations also showed high phenotypic resistance. Based on the presence of one or two resistance alleles that are linked to the Ren1 locus, 32 resistant seedlings and 41 resistant in situ genotypes were identified in the investigated set. Eight seedlings showed consistent phenotypic PM resistance, of which seven carried one or two alleles at the tested markers. This study provides the first evidence of PM resistance present within the eastern Adriatic V. sylvestris germplasm.

Full text

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Identication of powdery mildew
resistance in wild grapevine (Vitis
vinifera subsp. sylvestris Gmel
Hegi) from Croatia and Bosnia
and Herzegovina
Katarina Lukšić1, Goran Zdunić1*, Katarina Hančević1, Maja Žulj Mihaljević2, Ana Mucalo1,
Erika Maul3, Summaira Riaz4 & Ivan Pejić2,5
Wild grapevine (Vitis vinifera subsp. sylvestris) is widely recognized as an important source of
resistance or tolerance genes for diseases and environmental stresses. Recent studies revealed partial
resistance to powdery mildew (Erysiphe necator, PM) in V. sylvestris from Central Asia. Here, we report
resistance to PM of V. sylvestris collected from dierent regions of Croatia and in seedling populations
established from in situ V. sylvestris accessions. Ninety-one in situ individuals and 67 V. sylvestris
seedlings were evaluated for PM resistance according to OIV 455 descriptor. Three SSR markers (SC47-
18, SC8-071-0014, and UDV-124) linked to PM resistance locus Ren1 were used to decipher allelic
structure. Nine seedlings showed resistance in in vivo evaluations while leaf disk assays revealed three
PM-resistant accessions. One V. vinifera cultivar used as a control for PM evaluations also showed high
phenotypic resistance. Based on the presence of one or two resistance alleles that are linked to the
Ren1 locus, 32 resistant seedlings and 41 resistant in situ genotypes were identied in the investigated
set. Eight seedlings showed consistent phenotypic PM resistance, of which seven carried one or two
alleles at the tested markers. This study provides the rst evidence of PM resistance present within the
eastern Adriatic V. sylvestris germplasm.
Powdery mildew (Erysiphe necator) is an economically important fungal disease of grapevine. It has been a
continuous problem since its introduction from North America to Europe around 1845. In < 10years, E. neca-
tor became a problem in vineyards throughout the Mediterranean1. Shortly aer the onset of this disease in
Europe, inorganic fungicides and sulfur were used to control powdery mildew (PM). ere is a risk of the fun-
gus developing resistance to fungicides2. Chemical protection is applied ~ 10 times during the growing season,
making viticulture one of the largest fungicide consumers worldwide3. Fungicides can also cause undesirable
characteristics, such as o-avors in wine4. Modern researchers are developing revolutionary methods to control
PM, including exposure of the fungus to UV light at night, when the fungal defense system ‘turns o’5. Another
solution comes from the grapevine genome itself. Twelve loci from diverse grapevine species originating in
North America, Central Asia and China carry genes for plant defense against PM6. Loci such as Ren4 and Ren6
(Resistance to Erysiphe necator) provide complete resistance, not allowing the fungus to proliferate7.
European grapevine (Vitis vinifera subsp. vinifera; hereaer called V. vinifera) is susceptible to powdery
mildew, generally showing no resistance8. However, strong natural resistance to PM was rst identied in the
Central Asian V. vinifera cultivar (cv.) ‘Kishmish vatkana’9, which carries the major locus Ren1 on chromosome
13. e Ren1 locus has many advantages in breeding programs and mediates partial resistance to PM6, similar
to loci Ren3 and Ren910.
Research on PM resistance was recently broadened by including the wild Eurasian grapevine (Vitis vinifera
subsp. sylvestris Gmel Hegi; hereaer called V. sylvestris) into disease evaluations. V. sylvestris usually grows in
OPEN
1Institute for Adriatic Crops and Karst Reclamation, Put Duilova 11, 21 000 Split, Croatia. 2University of Zagreb,
Faculty of Agriculture, Svetošimunska cesta 25, 10 000 Zagreb, Croatia. 3Julius Kühn-Institute, Federal
Research Centre for Cultivated Plants, Institute for Grapevine Breeding Geilweilerhof, 76833 Siebeldingen,
Germany. 4Department of Viticulture and Enology, University of California, Davis, CA 95616, USA. 5Centre of
Excellence for Biodiversity and Molecular Plant Breeding, 10 000 Zagreb, Croatia. *email: goran.zdunic@krs.hr
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habitats isolated from human impact. It is mostly susceptible to PM11,12, but considerably less aected by the
disease than V. vinifera11,13 indicating some level of tolerance to PM11. V. sylvestris populations in northern Spain
were susceptible to PM12, as were vines growing in six river basins in southern Spain14, with varying infection
levels of dierent parasitic species in vines from the same location.
Recent identication of Ren1 in two V. sylvestris accessions from Central Asia revealed that PM resistance is
located at the same genetic position on chromosome 13 as in cv. ’Kishmish vatkana’15. Additionally, a few dozen
PM-resistant V. sylvestris accessions from Central Asia were identied as carriers of new resistance-linked alleles
at ve microsatellite (SSR) markers associated with the Ren1 locus7. e presence of Ren1 in V. sylvestris is very
intriguing for the grapevine breeding community. e genetic resources of V. sylvestris in European populations
represent important sources of genetic variability that are worth preserving. However, no research has investi-
gated the presence of resistance-linked alleles at the Ren1 locus.
V. sylvestris has been referenced in Croatian literature historically16,17. Recently, Croatian V. sylvestris was
systematically identied and characterized18–20 as belonging to ve natural populations in humid Mediterranean
forests and in sub-Mediterranean ecosystems19. Prospecting at natural sites is ongoing and recently two additional
populations were identied (data not published). However, these studies did not evaluate resistance to fungal
disease among the populations.
To bridge that gap, the present work (i) evaluates PM resistance of V. sylvestris accessions from the eastern
Adriatic region (Croatia and Bosnia and Herzegovina) using simple sequence repeat (SSR) markers linked to the
powdery mildew resistance locus Ren1, and (ii) performs a detailed phenotypic evaluation of visible powdery
mildew symptoms on V. sylvestris seedlings. is is the rst report of PM evaluations in V. sylvestris collected
in Croatia and Bosnia and Herzegovina and identies germplasm that could be used for grapevine breeding.
Results
Disease evaluations. e inventory of V. sylvestris in their natural habitats found very few vines with
powdery mildew (PM) symptoms. Only ~ 7% of individuals developed PM symptoms, mostly from the Krka
National Park population. Detailed evaluation for PM resistance continued on 67V. sylvestris seedlings grown
in pots in an ex-situ seedling collection.
Seedlings were previously established from seeds of ve female individuals during inventory work aiming at
gene conservation. Each seedling was represented by one biological replicate due to juvenile growth phase and
lower plant vigour in a shaded collection.
Visual PM symptoms on each accession were rated according to the OIV 455 descriptor using a ve-class
scale (1–9), for both invivo observations of the collection and invitro leaf disk testing (Fig.1).
Two-year invivo evaluation of PM on entire seedling plants was possible for 62 and 57 seedlings out of 67
seedlings in 2018 and 2019, respectively (Fig.2a). Failing accessions either dried up or were too small at the time
of observation. Nearly half of the seedlings analyzed in 2018 (30 accessions) showed intermediate PM resistance
(score 5). Twenty-two accessions were resistant: 15 resistant and seven very resistant (scores 7 and 9, respectively).
Nine accessions were susceptible and one was very susceptible to PM (scores 3 and 1, respectively). Similar trends
were observed in 2019. In both years, intermediate resistance was conrmed in 12 accessions, resistance in nine
and susceptibility in four (Table1). ree control accessions of V. vinifera cultivar ‘Plavac mali sivi’ showed resist-
ance to high resistance, except that accession PMS22 had intermediate resistance in 2018 (Fig.2a).
A subset of 35 seedlings with the best tness in 2018 and 2019 was chosen for the leaf disk assay. ree V.
vinifera cvs. were used as controls (Fig.2b). e majority of seedlings (20 accessions) in 2018 showed PM resist-
ance: 18 were resistant and two very resistant. Intermediate resistance was found in 11 accessions and four were
susceptible. In 2019, only ve accessions of the same subset were resistant, 17 accessions had intermediate resist-
ance, while 13 were susceptible. ree seedlings (SjCer14, SjCer23 and SjCer24) had leaf disk PM resistance in
both years. ree seedlings had intermediate resistance and one was susceptible in both years (Table1). Control
Figure1. Grapevine powdery mildew (E. necator) mycelium development on V. sylvestris leaves. Powdery
mildew spores were inoculated onto the top (adaxial) surface of leaf disks of V. sylvestris seedlings. Each gure
represents fungal growth on a single leaf disk of ve dierent genotypes according to the OIV 455 scale from
1 to 9 (le to right), respectively. e PM-susceptible seedlings received a lower scale number, while resistant
seedlings received a higher number.
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cv. ’Plavac mali sivi’ (PMS 39) showed intermediate resistance, ’Pošip bijeli’ (PSP 01 and PSP B2) displayed very
high resistance in 2018, but were susceptible and resistant, respectively, in 2019. ’Rukatac’ (RKTC 02) was rated
highly resistant in 2018 and very susceptible in 2019.
Genetic polymorphism at the Ren1 locus with reference to phenotypic disease evalua-
tion. Genetic SSR data based on three markers: SC47-18, SC8-0071-14 and UDV124, were adapted to match
alleles with previous studies7,15. irty-two out of 67 seedlings had the R-allele at one or two SSR markers
(Table1). R-allele 239 at marker SC47-18 was the most frequent (29 seedlings). Five seedlings had R-allele 212
at UDV124. Two seedlings had both 239 and 212 R-alleles.
Seedlings from the Cerovica population showed the greatest overall phenotypic resistance (mean value 6),
while seedlings from the Gizdavac population had the least overall resistance (4.97), despite relatively high
R-allele variability.
None of the seedlings showed full resistance, evaluated using two phenotypic approaches, over both years
(Table1). However, three seedlings were resistant when analyzed invivo and in one year of leaf disk assay:
SjPak13 (no R-alleles), SjCer7 (allele 212), and SjCer12 (allele 239). ree Cerovica seedlings, SjCer14, SjCer23,
and SjCer24, showed resistance in leaf disk assays and in one year of invivo. Seedling SjCer23 had two R-alleles
(239 and 212), SjCer14 one (239), and SjCer24 had none of the R-alleles.
PM resistance of in situ V. sylvestris genotypes. Ninety-one V. sylvestris individuals from natural
habitats (in situ) were analyzed at three SSR markers (Table2). Forty-one individuals carried alleles associated
with PM resistance. All these individuals had R-allele 239 at SC47-18 except one (Im4), which had R-allele 246
at the same marker. One accession (Luk8) contained both alleles: 239 and 246. e Paklenica population had the
most individuals carrying R-alleles (13), followed by Psunj (12) and Lukovdol (8). Populations Imotski (4), Krka
(2) and Gizdavac (1) had the fewest individuals with R-alleles.
None of the insitu-tested individuals had R-alleles at SC8-0071-014 and UDV124, despite polymorphisms
at both markers (Table2).
Figure2. Resistance of V. sylvestris seedlings to grapevine powdery mildew (E. necator) based on OIV 455
descriptor ve-class scale (1–9) and two phenotypic approaches: (a) Invivo evaluation in 2018 encompassed 62
accessions and 57 accessions in 2019. V. vinifera cv. ‘Plavac mali sivi’ (PMS) was used as control. (b) Leaf disk
assay on a subset of 35V. sylvestris accessions in 2018 and 2019. ree V. vinifera cvs. were used as controls. Each
chart column in (a,b) shows the number of dierent accessions sharing the same OIV score (level of resistance)
in the year of evaluation. Unless otherwise indicated, all accessions were represented by a single biological
replicate.
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Population Seedling
OIV455 score SSR markers at Ren1
In vivo Leaf disk UDV124 SC8-0071-
014SC47-18
2018 2019 2018 2019
Gizdavac SjGiz2 5 7 7 3 203 210 157 199 213 238
Gizdavac SjGiz7 9 5 5 3 201 210 157 163 213 233
Gizdavac SjGiz9 5 5 7 3 203 214 163 199 233 238
Gizdavac SjGiz10 9 5 7 5 185 203 163 199 233 238
Gizdavac SjGiz12 9 5 5 3 185 203 163 199 233 238
Gizdavac SjGiz14 5 3 5 5 203 212 159 199 234 238
Gizdavac SjGiz17 3 5 5 3 203 203 159 199 238 238
Gizdavac SjGiz18 5 5 9 5 203 210 157 199 238 239
Gizdavac SjGiz19 5 5 5 3 203 203 163 197 233 239
Gizdavac SjGiz20 5 5 5 5 185 203 199 199 238 238
Gizdavac SjGiz21 5 3 5 7 201 216 163 171 213 233
Gizdavac SjGiz22 3 3 7 5 201 212 159 163 233 234
Gizdavac SjGiz23 5 5 5 3 185 201 163 197 233 239
Gizdavac SjGiz24 3 3 7 3 185 203 197 199 238 239
Gizdavac SjGiz25 5 3 9 3 203 210 157 199 213 238
Paklenica SjPak9 5 7 7 5 201 225 157 199 238 238
Paklenica SjPak11 9 5 7 5 185 203 167 199 238 238
Paklenica SjPak12 7 3 7 5 185 203 167 199 238 238
Paklenica SjPak13 7 9 7 1 185 214 197 199 234 238
Paklenica SjPak16 7 5 7 5 185 203 167 199 238 238
Paklenica SjPak17 3 7 1 3 185 203 167 199 238 238
Cerovica SjCer6 7 9 5 3 203 214 159 197 238 239
Cerovica SjCer7 7 7 7 5 185 212 157 197 233 234
Cerovica SjCer8 7 7 5 5 185 203 159 197 238 239
Cerovica SjCer10 5 7 7 5 203 203 197 197 239 239
Cerovica SjCer11 5 5 3 5 185 214 159 197 233 238
Cerovica SjCer12 7 7 7 3 203 203 197 197 234 239
Cerovica SjCer13 5 7 3 5 203 203 197 197 239 239
Cerovica SjCer14 5 9 7 7 185 203 197 197 233 239
Cerovica SjCer17 5 5 5 7 203 203 159 197 234 239
Cerovica SjCer21 7 5 7 5 185 214 159 197 233 238
Cerovica SjCer23 5 9 7 7 203 212 197 197 239 239
Cerovica SjCer24 5 7 7 7 185 203 159 197 233 238
Cerovica SjCer25 5 3 7 5 185 203 197 197 233 239
Cerovica SjCer26 3 3 3 5 185 203 197 197 233 239
Gizdavac SjGiz1 7 5 201 203 163 199 233 238
Table 1. (continued)
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Table 1. Genetic polymorphism of 67V. sylvestris seedlings at three SSR markers linked to the Ren1 locus on
chromosome 13 and phenotypic powdery mildew results obtained from invivo and leaf disk methods based
on the OIV 455 descriptor scale from 1 (susceptible) to 9 (resistant) in 2018 and 2019. Dashed line separates
seedlings based on phenotypic method used for their disease evaluation. For 10 seedlings, phenotypic results
were not determined or not complete (‘nd’) as accessions dried or were too small for evaluation. Rows in italics
individuals carrying R-alleles. Allele 239 at marker SC47-18 (in bold) was linked to resistance in V. sylvestris.
Allele 212 at marker UDV124 (in bold) was linked to resistance in V. vinifera7.
Gizdavac SjGiz3 5 5 203 214 199 199 238 238
Gizdavac SjGiz4 5 5 201 203 163 163 233 233
Gizdavac SjGiz5 7 5 185 201 163 163 233 233
Gizdavac SjGiz6 5 5 185 203 199 203 233 238
Gizdavac SjGiz8 nd nd 201 210 159 163 233 238
Gizdavac SjGiz11 nd nd 201 210 159 163 233 238
Gizdavac SjGiz13 1 nd 201 214 163 197 233 238
Gizdavac SjGiz15 5 5 201 203 163 197 233 239
Gizdavac SjGiz16 5 3 185 201 163 163 233 233
Paklenica SjPak1 nd nd 185 203 167 197 238 239
Paklenica SjPak2 nd nd 201 203 159 167 234 238
Paklenica SjPak3 9 7 185 203 167 167 238 239
Paklenica SjPak4 3 5 201 203 167 197 234 238
Paklenica SjPak5 9 nd 225 225 157 199 238 238
Paklenica SjPak10 5 nd 185 203 199 199 234 238
Paklenica SjPak14 nd nd 185 214 197 199 234 238
Paklenica SjPak15 3 3 185 203 167 199 238 238
Paklenica SjPak18 3 nd 185 203 197 199 237 238
Cerovica SjCer1 9 nd 185 212 197 197 233 239
Cerovica SjCer2 7 9 203 214 197 197 239 239
Cerovica SjCer3 5 7 203 203 159 197 238 239
Cerovica SjCer4 5 9 203 203 159 197 238 239
Cerovica SjCer5 5 7 185 203 197 197 233 239
Cerovica SjCer9 5 5 203 214 159 197 238 239
Cerovica SjCer15 7 7 203 203 197 197 239 239
Cerovica SjCer16 5 9 185 214 159 197 233 238
Cerovica SjCer18 7 7 185 203 197 197 233 239
Cerovica SjCer19 3 9 203 203 197 197 234 239
Cerovica SjCer20 5 9 185 203 197 197 239 239
Cerovica SjCer22 7 5 203 203 159 197 234 239
Cerovica SjCer27 7 5 201 203 163 197 233 239
Population Seedling
OIV455 score SSR markers at Ren1
In vivo Leaf disk UDV124SC8-0071-
014SC47-18
2018 2019 2018 2019
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Population Genotype
SSR markers at Ren1
UDV124 SC8-0071-
014 SC47-18
Paklenica Pak1 203 225 157 159 234 238
Paklenica Pak2 201 203 167 167 238 238
Paklenica Pak3 201 225 157 167 238 238
Paklenica Pak5 201 225 157 167 228 238
Paklenica Pak6 185 201 167 197 234 234
Paklenica Pak7 185 201 197 197 239 239
Paklenica Pak8 201 203 167 199 234 239
Paklenica Pak9 185 201 163 197 233 239
Paklenica Pak10 185 225 167 167 228 238
Paklenica Pak11 203 203 167 197 238 239
Paklenica Pak12 203 225 157 167 238 239
Paklenica Pak13 185 225 157 197 238 239
Paklenica Pak14 183 225 157 157 238 238
Paklenica Pak15 201 225 197 199 238 239
Paklenica Pak16 201 201 159 199 234 238
Paklenica Pak17 201 225 159 203 238 238
Paklenica Pak18 185 201 197 199 234 238
Paklenica Pak19 183 185 157 167 238 244
Paklenica Pak20 183 201 157 197 239 244
Paklenica Pak21 201 203 167 199 238 238
Paklenica Pak22 185 201 159 197 238 239
Paklenica Pak23 185 201 159 197 238 239
Paklenica Pak24 185 203 163 197 233 239
Paklenica Pak25 183 185 157 197 239 244
Paklenica Pak26 185 185 161 197 220 234
Paklenica Pak27 183 201 157 167 233 238
Paklenica Pak28 185 203 167 197 234 238
Paklenica Pak29 185 214 199 199 238 238
Paklenica Pak30 201 225 163 197 233 239
Paklenica Pak32 201 203 167 199 238 238
Paklenica Pak33 201 203 167 199 228 238
Paklenica Pak34 203 225 157 199 238 238
Imotski Im3 203 203 161 199 220 238
Imotski Im4 203 214 163 167 233 246
Imotski Im5 185 214 159 163 238 238
Imotski Im7 185 203 197 197 239 239
Imotski Im8 183 214 157 159 238 238
Imotski Im11 203 214 159 167 228 238
Imotski Im14 185 214 159 197 238 239
Imotski Im17 203 214 159 161 220 238
Imotski Im18 203 214 161 163 220 233
Imotski Im19 203 214 163 199 233 238
Imotski Im20 214 214 159 159 238 238
Imotski Im21 203 203 157 197 228 239
Lukovdol Luk1 201 214 159 159 238 238
Lukovdol Luk2 203 225 161 203 220 239
Lukovdol Luk3 201 203 159 167 238 244
Lukovdol Luk4 201 203 167 197 238 238
Lukovdol Luk5 201 203 161 197 234 239
Lukovdol Luk6 201 203 167 167 238 238
Lukovdol Luk8 201 225 159 197 239 246
Lukovdol Luk10 201 203 159 197 238 239
Lukovdol Luk11 201 203 197 203 238 239
Lukovdol Luk12 203 203 159 203 238 238
Lukovdol Luk13 201 203 197 203 233 238
Continued
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e set of 91 insitu-tested individuals was subjected to neighbor-joining (NJ) clustering and perceptual
mapping (Principal Coordinate Analysis, PCoA) to visualize the similarity among individuals based on SSR
allelic proles (Fig.3a,b). e NJ tree showed clusters of mixed individuals from dierent populations, only
roughly outlining their geographical origin. However, individuals with R-alleles are clearly more abundant on
the right side of the tree (red dots, Fig.3a). Relative relationships between individuals based on PCoA showed the
same pattern as NJ. ey separated individuals with R- alleles on the right side of plot from individuals without
R-alleles at Ren1 on the le side. PCoA projections of the rst two dimensions accounted for 33.69% of the total
molecular variation. PCoA revealed slight overlapping between the two groups (Fig.3b).
Genetic diversity. Genetic diversity was calculated for the two sets of V. sylvestris: 91 insitu individuals
and 67 seedlings. ree SSR markers were polymorphic in both sets (Table3). e observed heterozygosity was
lower than the expected heterozygosity, except at marker UDV124 and for the insitu set at marker SC47-18.
e insitu set showed more heterozygosity at the tested markers than the seedling set. e allele frequency (AF)
for resistance-related allele 212 at UDV124 was determined only in the seedling set with (AF = 0.04). At marker
SC8-0071-014, no resistance-linked alleles were observed. e AF was lower than 0.3 for all alleles except for the
Table 2. Genetic polymorphism of 91 insitu-tested V. sylvestris individuals at three SSR markers linked to the
Ren1 locus on chromosome 13. Rows in italics individuals carrying R-alleles. Allele 239 at marker SC47-18 (in
bold) was linked to resistance in V. sylvestris. Allele 246 at marker SC47-18 (in bold) was previously linked to
resistance in V. vinifera7.
Population Genotype
SSR markers at Ren1
UDV124 SC8-0071-
014 SC47-18
Lukovdol Luk14 201 214 197 199 234 238
Lukovdol Luk15 201 214 159 159 238 239
Lukovdol Luk16 201 203 199 203 233 238
Lukovdol Luk17 201 203 167 167 233 233
Lukovdol Luk18 203 214 159 167 238 239
Lukovdol Luk19 201 201 159 197 233 238
Lukovdol Luk20 201 201 159 197 238 239
Grab Grab1 185 203 163 197 233 239
Krka Krka9 203 214 157 167 238 239
Krka Krka15 185 201 197 197 234 234
Krka Krka18 185 203 161 197 220 234
Krka Krka19 203 203 161 197 220 234
Krka Krka20 185 203 197 197 234 239
Krka Krka21 203 203 161 197 220 234
Krka Krka24 185 185 161 167 220 238
Krka Krka26 201 203 197 197 234 234
Krka Krka27 185 203 161 197 220 234
Psunj Psunj3 203 214 167 199 234 238
Psunj Psunj4 201 227 195 197 238 239
Psunj Psunj5 201 201 161 167 220 238
Psunj Psunj7 203 214 159 161 220 238
Psunj Psunj8 201 203 167 197 238 239
Psunj Psunj10 203 214 167 199 238 239
Psunj Psunj11 203 214 197 197 239 239
Psunj Psunj12 203 214 197 197 239 239
Psunj Psunj14 201 214 167 199 238 239
Psunj Psunj21 201 203 163 167 233 238
Psunj Psunj22 201 214 197 199 238 239
Psunj Psunj23 193 214 159 161 220 239
Psunj Psunj24 201 201 201 203 239 239
Psunj Psunj25 193 214 159 161 220 239
Psunj Psunj26 193 214 159 161 220 239
Psunj Psunj27 193 201 161 167 220 238
Psunj Psunj28 214 214 159 167 238 239
Gizdavac Giz1 201 203 163 199 233 238
Gizdavac Giz2 185 203 163 197 233 239
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197 allele in seedlings, which was present in the entire Cerovica set (Table1). e greatest abundance of R-alleles
was found at marker SC47-18: R-allele 239 was found in the insitu and seedling sets at an AF of 0.25 and 0.26,
respectively. An additional R-allele 246 was observed in the insitu set only, at a very low AF of 0.01.
Discussion
is study represents the rst screening for powdery mildew resistance in eastern Adriatic V. sylvestris germ-
plasm. e phenotypic focus of study was on the V. sylvestris seedlings in an ex situ collection. ese were young
potted plants evaluated for powdery mildew resistance under invivo and invitro conditions. e resistance
level of seedlings varied from very susceptible to medium to highly resistant. e control cultivars in this study
showed medium to very high resistance (OIV455 scores from 5 to 9), except cvs. ‘Rukatac’ and ‘Pošip bijeli’ in
2019. ese white grape cultivars are considered susceptible to fungal diseases. None of the control cultivars
had R-alleles at the three SSR markers, even though cv. ‘Plavac mali sivi’ is known to have good tolerance toward
fungal infections. Control cultivars clearly expressed more robust vigour than V. sylvestris accessions. Twenty-two
and 23 invivo-evaluated seedlings showed high resistance to PM in 2018 and 2019, respectively. e majority of
seedlings showed PM infection, indicating homogeneous eld infections. Seedlings from the Cerovica popula-
tion (Bosnia and Herzegovina; Fig.4, Table1) had the greatest average resistance and the most accessions with
R-alleles (23 out of 27 accessions). Cerovica, which is part of the large Neretva population in Bosnia and Her-
zegovina, contained a considerable number of dierent and private alleles20 and possibly retained an important
fraction of the historic V. sylvestris biodiversity. e seedlings from Paklenica and Gizdavac had intermediate
resistance. e Paklenica population is one of the most-conserved V. sylvestris populations in Croatia and had the
most R-alleles in its insitu accessions, while Gizdavac is one of the most vulnerable populations, close to urban
centers and with visible human impact19,23. Seedlings from Gizdavac had greater allelic diversity of SSR markers
than those from Paklenica (the least), even though the overall phenotypic resistance of Gizdavac seedlings was
the lowest in this study.
During invivo evaluation, nine seedlings were conrmed as resistant in both years. Of these nine, three were
susceptible in the leaf disk assay. Seedling SjCer7 had the greatest leaf disk resistance and carried R-allele 212
at marker UDV124. is allele is associated with resistance in V. vinifera 6. On the other hand, four seedlings
were susceptible both years invivo. One had a single R-allele 212, two had a single R-allele 239 and one had no
R-alleles.
PM resistance (score 7) in the seedling subset leaf disk assay was conrmed in both years for only three
accessions from the Cerovica population. e resistant accessions SjCer14, carrying the single allele 239, and
SjCer23, carrying two alleles (212 and 239), had the same phenotypic disease scores regardless of the evaluation
method and were slightly more resistant than SjCer24, the resistant accession without R-alleles. Among the entire
seedling set, only two accessions from Cerovica, SjCer1 and SjCer23, had R-alleles at two markers (UDV124 and
SC47-18) and expressed PM resistance.
In general, disease evaluations are very subjective and vary among studies due to dierent environmental
conditions, experimental set up, disease pressure, and pathogen population structure. e phenotypic evaluations
Figure3. Clustering of 91 wild grapevine individuals based on three SSR markers at the Ren1 locus. (a)
Neighbor-joining dendrogram showing genetic relationships among individuals with bootstrap support
value ≥ 75%. e samples with resistance alleles are marked with red dots. Analyses were conducted in
MEGA721. (b) Principal coordinate (PCoA) projections of individuals in a 2-D plot based on genotypic
distances conducted in GenAlex22.
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conducted in this study diered to some extent from Riaz etal.7, in which a modied and inverted OIV scale
was used. Similar modications were reported in other studies8,10,24,25. e seedling plants in this study were very
young at the time of phenotypic evaluations, which probably aected both the level of resistance and disease
pressure to some extent. Young plants do not have excess vegetative growth, which allows more air circulation
and potentially reduces infection severity. Nevertheless, the nal outcomes were comparable. In this study, most
seedlings showed intermediate resistance to PM, characterized by clearly visible, but localized and fragmented,
sporulation with small patches of mycelium. However, in the 2018 invitro analysis, most seedlings expressed
resistance to PM. Mycelial growth was highly suppressed and conidiospores were not observed. e resistance of
the seedling set under invitro conditions varied greatly between the two seasons (14 and 57% of the accessions
expressed resistance). Under invivo conditions, unsprayed, naturally-infected plants showed more consistency
between seasons, with the number of resistant seedlings varying between 35 and 40%. is variation might be
because of using more mature leaves in the assay and variation in the PM inoculum, which was collected from
the eld. Field PM strains can vary from one year to another26, which might cause variations in resistance levels.
In our samples, intermediate resistance was predominant. is diered from phenotyping results of Riaz etal.7,
where the intermediate rating was not observed among the seedlings. is was probably due to more consistent
invitro assay infections and inoculation with a single PM strain collected from pure cultures7. In our study,
randomly-collected, spontaneous eld PM inoculum was used. Moreover, full parentage was known for only nine
seedlings (23% of the seedling set), thus information on the phenotypic resistance to PM of the insitu parental
lineages is entirely missing. In a previous study7, the F1 population of seedlings resulted from crosses between
Table 3. Genetic diversity at three SSR markers linked to the Ren1 locus for powdery mildew resistance in V.
sylvestris. e alleles in bold are linked to resistance at Ren1 according to Riaz etal.7,15. ’Na’ is the number of
dierent alleles, ’Ho’ is the observed heterozygosity, and ’He’ is the expected heterozygosity.
Locus Na Ho He Allele
Allele frequency
In situ V. sylvestris V. sylvestris seedlings
UDV124
In situ
V. sylvestris 8 0.84 0.79 183 0.03
V. sylvestris seedlings 8 0.82 0.72 185 0.14 0.24
193 0.02
201 0.28 0.12
203 0.30 0.45
210 0.05
212 0.04
214 0.15 0.08
216 0.01
225 0.07 0.02
227 0.01
SC8-0071-014
In situ
V. sylvestris 11 0.82 0.84 157 0.08 0.05
V. sylvestris seedlings 8 0.69 0.77 159 0.15 0.13
161 0.09
163 0.06 0.15
166 0.01
167 0.19 0.08
171 0.01
195 0.01
197 0.26 0.38
199 0.10 0.20
201 0.01
203 0.04 0.01
SC47-18
In situ
V. sylvestris 8 0.75 0.75 213 0.03
V. sylvestris seedlings 220 0.09
6 0.72 0.73 228 0.03
233 0.09 0.24
234 0.11 0.09
237 0.01
238 0.41 0.37
239 0.25 0.26
244 0.02
246 0.01
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known resistant (V. sylvestris) and susceptible (V. vinifera) parents. Research on ‘omics’ provides additional and
unique insight into the linking of phenotypes with genotype interactions and plant stress responses27. Com-
prehensive research on the transcriptomics of Central Asian accessions revealed varying levels of phenotypic
resistance to PM, matched with various transcriptomic responses to E. necator among Central Asian accessions24.
e three Ren1-linked SSR markers on chromosome 13 that we analyzed insitu in Croatian V. sylvestris
populations and in seedlings were highly polymorphic. e observed heterozygosity was lower than the expected
heterozygosity, indicating lower genetic diversity among the studied sets at the three SSR markers. is is simi-
lar to what Riaz etal.7 found at four (of ve) R-SSR loci for the Central Asian grape germplasm, pointing to
inbreeding due to geographic isolation of the populations. Increased gene ow might be assumed, for instance,
in seedlings from Gizdavac, where out of two individuals, only the male parent, Giz2, had the R-allele 239, which
was not found in its progeny (SjGiz4). Eight seedlings from Gizdavac had various R-alleles that were most likely
inherited from their unknown paternal parents. e Gizdavac population, near urban centers, diers from the
protected Paklenica population, where most insitu individuals had an R allele. Only two progeny seedlings had
an R-allele, 239, which they shared with their mother accession, Pak12. SNP Genotyping by Sequencing approach
revealed signicant and unexpectedly high segregation distortions from Mendelian ratios on chromosome 13 in
the susceptible V. vinifera cv. ‘Glera’, making one end of its chromatide less-inherited in the ospring28.
In this study, none of the accessions had R-alleles (141 or 143) at the SC8-0071-14 locus. is locus was
previously shown to be polymorphic and a signicant Quantitative trait loci (QTL), explaining up to 96% of the
variation. It was mapped together with the SC47-18 locus. In a previous study7, SC8-0071-014 R-alleles were
detected in both resistant and susceptible accessions. Here, accessions carrying R-alleles at UDV124 and SC47-18
were either resistant or susceptible. Locus UDV124 anks Ren1 on chromosome 13, while loci SC8-0071–014 and
SC47-18 co-segregated with resistance (Ren1)29. Clustering by NJ and PCoA both separated individuals carrying
R-alleles to the right side of the graphs (Fig.3a,b). ere was no clear phylogenetic separation of individuals by
population of origin, although there was rough separation between northern and southern populations (Fig.3a).
PCoA more clearly visualized the overlapp between two groups. ese results may indicate a common source of
Ren1 loci in studied V. sylvestris. However, as was recently discussed for Caucasus V. vinifera cvs., high complex-
ity at the chromosome 13 region and information gathered thus far does not permit conclusions as to whether
studied genotypes from the Caucasus and Central Asia share the same resistance genes28.
is study provides insight into powdery mildew resistance of V. sylvestris accessions from the eastern Adri-
atic region. e observed phenotypic resistance of V. sylvestris individuals to powdery mildew was clear and
Figure4. Map of seven insitu Croatian V. sylvestris populations and the Cerovica population in Bosnia and
Herzegovina.
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consistent for some individuals, but showed some weakness in connecting phenotypic and genetic resistance in
other individuals. e SC47-18 marker (R-allele 239) co-segregated with resistance at Ren1. It was the most domi-
nant marker in our V. sylvestris set and there were no R-alleles at another co-segregating marker, SC8-0071-014.
However, a trend with two new allelic combinations was found in the studied set. is has interest for a deeper
investigation on functional PM resistance related to that marker and the Ren1 locus. No substantial connection
was observed between individuals with greater phenotypic resistance and expected resistant genotypes. is
result was in accordance with previous results, in which both resistant and susceptible genotypes had R-alleles.
However, accessions with two R-alleles at two dierent SSR markers showed, on average, greater resistance to
powdery mildew in this study. e presence of R-alleles in the eastern Adriatic V. sylvestris conrms these genetic
resources as important sources of biodiversity. Next-generation sequencing technologies and other –omics meth-
ods would be benecial to access more information on the nature of PM resistance in the eastern Adriatic region.
Methods
Plant material. A total of 158 unique V. sylvestris genotypes were analyzed in this study. e sample set con-
sisted of 91V. sylvestris genotypes from seven natural populations in Croatia (in situ) and 67V. sylvestris seed-
lings established from seeds of ve V. sylvestris plants from three natural populations: Paklenica and Gizdavac in
Croatia and Cerovica in Bosnia-Herzegovina. All V. sylvestris individuals included in this study were identied
morphologically and using 20 SSR markers through our previous inventorying of V. sylvestris in Croatia and
neighboring countries18,20,30. Permission to collect and examine the plant species Vitis vinifera subsp. sylvestris
within protected natural areas of Croatia has been granted by the Ministry of Economy and Sustainable Develop-
ment of the Republic of Croatia (Permission No. UP/I-612-07/15-33/74). e voucher specimens were identied
by Goran Zdunić and Katarina Lukšić and are deposited in the publicly accessible herbarium of the Institute for
Adriatic Crops and Karst Reclamation, Split, Croatia.
e natural habitats of the studied species cover diverse geographic areas: the eastern coast of the Adriatic
Sea, the mountainous area of the Dinaric Alps, and the mountainous area in the Croatian part of the Pannonian
Basin. Supplementary TableS1 lists the analyzed genotypes, their geographical origins and their sampling loca-
tions. e seven natural V. sylvestris populations were from Imotski, Grab, Gizdavac, Krka, Paklenica, Lukovdol
and Psunj (Fig.4).
Disease evaluations. PM symptoms were rst observed on wild individuals insitu during an inventory of
V. sylvestris around bloom. e overall health status of each plant was evaluated. PM symptoms on green tissues,
including leaves (adaxial surfaces), were noted: white to grayish coatings of fungal colonies, upward leaf curling,
and leaves that were drying and falling o.
e insitu V. sylvestris plants were not subjected to leaf disk assays in this study due to their spatial dislocation
and the diculty of keeping the plant material fresh and ensuring uniform trial conditions.
PM evaluation of the 67V. sylvestris seedlings was performed (with no biological replicates) on plants and
via a leaf disk assay (only 35 seedlings) using the OIV 455 descriptor31. Each genotype was evaluated by visual
inspection for signs of pathogens using a stereomicroscope and the OIV 455 scale: 1 = unlimited infection; com-
plete or nearly complete attack of the leave surface, abundant mycelial growth 3 = vast attacked patches, some of
which were limited, obvious mycelial growth and fungus fructication; 5 = attacked patches were frequent, but
usually clearly limited; 7 = sparse, small and limited attacked patches; little mycelium and fungus fructication,
9 = greatly suppressed symptoms or none at all; no mycelium or visible fructication.
e seedlings were grown in pots in an outdoor shaded area and were not sprayed from the beginning of
vegetation, before and during the PM evaluations. e disease evaluations were conducted during 2018 and 2019.
e subset of 35 out of 67 seedlings selected for the invitro leaf disk assays included only individuals with
the best health status and overall tness.
e invivo PM evaluations were performed on May 21st in 2018 and on May 13th in 2019, during the highest
disease pressure in the eld collection period. e observations encompassed 67V. sylvestris seedlings and the
cv. ’Plavac mali sivi’ as a control with three accessions. e invivo inspections were carried out by evaluating
an approximately equal part of each plant and taking into consideration the overall plant health, aer which a
single OIV 455 score was assigned per genotype.
For the leaf disk analyses, besides 35V. sylvestris samples (Table1), four potted cvs. ’Plavac mali sivi’ (1 rep-
licate), ’Pošip bijeli’ (2 replicates) and ’Rukatac’ (1 replicate), were included as references. e pink cv. ’Plavac
mali sivi’ is generally known to be less susceptible to mildew, while the white cvs. ’Rukatac’ and ’Pošip bijeli’ are
susceptible.
Leaf disk assays. e invitro analyses were performed by placing leaf disks on 1% water-agar (Bacto™
Agar, BD, France) in plastic Petri dishes, as described8,32. e leaf disks were infected with E. necator using fresh
conidia from naturally infected leaves of susceptible V. sylvestris seedlings grown in the collection. e spores
were collected with a small so brush and transferred onto each adaxial leaf surface. e fourth to sixth fully
developed leaves of each genotype were sampled: two leaves (four leaf disks) were analyzed per sample. e sam-
ples were placed in a climate chamber at 25°C and 65% relative humidity on a 16-h light, 8-h dark cycle. Seven to
nine days post inoculation (dpi), leaves were evaluated for mycelial growth and conidiophore formation. Evalu-
ations of the infections were performed using a CarlZeiss stereomicroscope (Microimaging, GmbH Germany),
and detailed inspections together with photos were captured under 0.65 × magnication.
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DNA extraction and marker analysis. DNA was extracted from the young leaves using the NucleoSpin
Plant II kit (Macherey–Nagel, Düren, Germany). e extracted DNA was quantied and used at a working DNA
concentration of 1ng/μL.
ree microsatellite markers, SC47-18, SC8–0071–14 and UDV1246, that are associated with the Ren1 gene
for PM resistance were analyzed. e SSR markers were multiplexed within one run. All forward primers were
labeled on the 5’ end with uorescent dyes (NED, 6-FAM and VIC).
A KAPA Fast Multiplex PCR Kit (2x) (Kapa Biosystems, Wilmington, MA) was used to set up 5-µL volume
reaction mixtures containing a master mix, 100pmol of each primer, and ~ 1ng template DNA. PCR amplica-
tion was performed using the following program: three minutes of initial denaturation at 95°C, followed by 30
cycles of denaturation at 95°C (15s), annealing at 60°C (30s), and extension at 72°C (30s). A nal extension
was performed at 72°C for seven min.
e amplied products were resolved using capillary electrophoresis on an ABI 3730xl Genetic Analyzer
(Applied Biosystems) using GeneScan-LIZ 500 as an internal standard. e peaks were identied with GeneMap-
per 4.0 soware (Applied Biosystems). e allele calling was harmonized with previously published SSR data7,15
by amplifying two identical V. sylvestris samples separately at the University of California at Davis and at the
Laboratory of the IAC in Split. en, the allele sizes were aligned among the other samples.
e trueness to type of the V. sylvestris set was analyzed and discussed in a previous study30.
Data analysis. e phenotypic data and corresponding statistical analyses were performed in Excel 2013.
e disease scores were determined according to the OIV 455 scale: by calculating mode values from four leaf
disk scores per genotype each year and by determining a direct, single OIV score per genotype for all invivo
evaluations each year.
Descriptive statistics of the SSR markers used in this study were calculated for the following indices: the
number of dierent alleles per locus (Na), expected heterozygosity (He), observed heterozygosity (Ho) and allele
frequency (AF). ese statistics were obtained using the GenAlEx 6.5 package22. Principal coordinate analysis
(PCoA) was performed to visualize relationships between individuals based on the Ren1 scoring data via covari-
ance, standardized using the same program.
e SSR data for 91 insitu individuals were analyzed to compute their evolutionary history using the Neigh-
bor-Joining method33 implemented in MEGA 7.0 soware21. e bootstrap interior branch test was used to test
the reliability of each interior branch on the tree34.
Data availability
All data generated or analyzed during this study are included in this published article (and its Supplementary
Information les). Correspondence and requests for materials should be addressed to G.Z. e study complies
with local and national regulations.
Received: 16 July 2021; Accepted: 20 January 2022
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Acknowledgements
We gratefully acknowledge the funding support of the Croatian Science Foundation (grant: UIP-2014-09-9737
and “Young researchers’ career development—training new doctoral students”) and the European Regional Fund
(grant KK.05.1.1.02.0010).
Author contributions
K.L., G.Z. and I.P. designed research; K.L. carried out phenotyping and genotyping of germplasm; K.H., M.Ž.M.
and A.M. performed research; E.M. and S.R. advised, reviewed and edited paper; K.L. and G.Z. wrote the paper.
All authors read and approved the nal manuscript.
Competing interests
e authors declare no competing interests.
Additional information
Supplementary Information e online version contains supplementary material available at https:// doi. org/
10. 1038/ s41598- 022- 06037-6.
Correspondence and requests for materials should be addressed to G.Z.
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