Abstract HOLLEINOVÁ, V., ČECHOVÁ, J.: The detection of viruses and phytoplasmas in dwarfed shoots of grapevine varieties The survey of occurrence of six chosen virus pathogens and phytoplasmas complex was done on plants of grapevine (Vitis vinifera L.) which showed symptoms of short-shoot syndrom. The results of serological and molecular tests did not confi rm either virus or phytoplasma infection as the main source of short-shoot syndrom. The presence of 6 viruses in samples taken from 45 aff ected plants of grapevine on 4 habitats. The highest occurrence of viruses was found out on habitat Moravská Nová Ves, where all taken samples were infected by Grapevine leafroll-associated virus GLRaV-1 (100 %). In 66 % of the samples taken from that habitat were detected mixed infection of Grapevine virus A and GLRaV-1. These 2 pathogens but were not detected in samples from aff ected plants from the other habitats or very sporadically -just in 2 plants. Another 4 virus pathogens were detected either sporadically (Grapevine fl eck virus, Grapevine leafroll-assoc. virus GLRaV-3, Arabis mosaic virus) or not at all (Grapevine fanleaf virus). From 270 tests made to 6 viruses were only 20 positive, e. g. 7.4 %. It means that from 45 plants were 15 infected at least by 1 virus (33 %). The phytoplasmas complex was tested in 28 plants. The result was positive only in 1 plant, by another test the Potato stolbur phytoplasma was confi rmed

J Čechová

V Holleinová

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ACTA UNIVERSITATIS AGRICULTURAE ET SILVICULTURAE MENDELIANAE BRUNENSIS
Volume LX 8 Number 8, 2012
THE DETECTION OF VIRUSES AND 
PHYTOPLASMAS IN DWARFED 
SHOOTS OF GRAPEVINE VARIETIES 
AURELIUS AND NEUBURGER
V. Holleinová, J. Čechová
Received: September 13, 2012
Abstract
HOLLEINOVÁ, V., ČECHOVÁ, J.: The detection of viruses and phytoplasmas in dwarfed shoots of grapevine 
varieties Aurelius and Neuburger.  Acta univ. agric. et silvic. Mendel. Brun., 2012, LX, No. 8, pp. 73–78
The survey of occurrence of six chosen virus pathogens and phytoplasmas complex was done on 
plants of grapevine (Vitis vinifera L.) which showed symptoms of short-shoot syndrom. The results 
of serological and molecular tests did not confi rm either virus or phytoplasma infection as the main 
source of short-shoot syndrom. The presence of 6 viruses in samples taken from 45 aff ected plants of 
grapevine on 4 habitats. The highest occurrence of viruses was found out on habitat Moravská Nová 
Ves, where all taken samples were infected by Grapevine leafroll-associated virus GLRaV-1 (100 %). In 66 % 
of the samples taken from that habitat were detected mixed infection of Grapevine virus A and GLRaV-1. 
These 2 pathogens but were not detected in samples from aff ected plants from the other habitats 
or very sporadically – just in 2 plants. Another 4 virus pathogens were detected either sporadically 
(Grapevine fl eck virus, Grapevine leafroll-assoc. virus GLRaV-3, Arabis mosaic virus) or not at all (Grapevine 
fanleaf virus). From 270 tests made to 6 viruses were only 20 positive, e. g. 7.4 %. It means that from 45 
plants were 15 infected at least by 1 virus (33 %). The phytoplasmas complex was tested in 28 plants. 
The result was positive only in 1 plant, by another test the Potato stolbur phytoplasma was confi rmed.  
ELISA, nested-PCR, symptoms, shoot, Vitis 
The research for presence of viruses in plants 
which were aff ected by growth disfunctions and 
harvest decrease of grapevine started in laboratory 
for diagnostic of plant viruses and phytoplasmas 
in institut Mendeleum in 2003. The growers 
pointed out the shortened growth of internodias 
which caused zig-zag growth of shoots (Fig. 1), low 
crop, and shortened clusters and stalks (Fig. 2). 
These symptoms were noticed mainly by varieties 
Neuburger and Aurelius and later by Sauvignon 
blanc and Saint Laurent. During the years with low 
precipitation and high temperatures during spring 
and summer was shortened growth of shoots very 
signifi cant, bunch of grapes minimised and their 
development limited.
Bovey et al. (1980) describe short internodia and 
harvest decrease as a symptom of virus infection 
by Grapevine fanleaf virus (GFLV) and Arabis mosaic 
virus (ArMV). The detection of these viruses by 
serological method DAS-ELISA in 2003–2009 did 
not confi rm defi nitely that these viruses caused 
that growth defect, in oposite – in some plants with 
strong symptoms these viruses were not detected 
at all. That’s why was the spectrum of tested 
viruses broaden for another 4 virus pathogens and 
phytoplasmas complex. Canadian Food Inspection 
Agency mentions that by phytoplasma Grapevine 
yellows diseases can appear tight clustering of young 
shoots, zig-zag growth and early rolling of leaves. 
Also can be noticed bad development of fl owers 
and fl ower stalk (2006). The occurrence of stolbur 
phytoplasma in grapevine was offi  cially confi rmed 
in Czech Republic by diagnostics laboratory of State 
Phytosanitary Administration in 2006 at varieties 
74 V. Holleinová, J. Čechová
Lemberger (Blaufränkisch), Zweigeltrebe and Saint 
Laurent in districts Břeclav, Znojmo and Brno 
(Červená et al., 2007).
The spread of rust mite (Calepitrimerus vitis Nal.) 
and bud mite (Colomerus vitis Pag.) in the vineyards 
is considered as the source of short shoot syndrome 
in Australia (Bernard et al., 2005) and also in Oregon 
and Washington (Walton et al., 2007).
MATERIAL AND METHODS
The samples of dormant shoots were taken from 
vineyards in 4 habitats in South Moravia from 
plants, which showed above mentioned symptoms 
of growth abnormalities. They were tested by 
serological method DAS- ELISA (Clark & Adams, 
1977) fi rst to presence of GFLV and ArMV viruses 
and then to Grapevine leafroll complex GLRaV-1 
and GLRaV-3, Grapevine fl eck virus and Grapevine 
1: Significant shortening of internodia on grapevine shoot 
2: Dwarfed claster
I: Overview of habitats and tested varieties
Habitat Variety Year of testing
Čejkovice Aurelius 2009, 2010
Dolní Dunajovice Aurelius, Neuburger 2010
Dolní Kounice Neuburger 2005, 2010
Moravská Nová Ves Neuburger 2010
 The detection of viruses and phytoplasmas in dwarfed shoots of grapevine varieties Aurelius and Neuburger 75
virus A. The diagnostics of Bioreba company 
(Switzerland) were used. The methodology was 
followed according to the producer’s instructions. 
The list of habitats and varieties is in the Tab. I.
The presence of phytoplasmas was tested by 
molecular method nested-PCR (polymerase chain 
reaction) from the sample, which contained mix 
of tissues of leaf, petiole with veins of 1st line and 
phloem from summer shoots. Isolation of DNA was 
made by modifi ed protocol (Ahrens & Seemüller, 
1992). In the fi rst PCR reaction were used primers 
P1/P7, in the second PCR reaction were used primers 
R16F2n/R16R2 (Gundersen & Lee, 1996). The samples 
were later separated by electrophoresis.
Positive samples from this test to phytoplasmas 
complex were then tested on presence of Potato 
stolbur phytoplasma. In this test were used 
following primer combinations: fi rst PCR reaction 
– STOL11F2/STOL11R1 (Daire et al., 1997) and second 
PCR reaction – STOL11F3/STOL11R2 (Clair et al., 
2003). 
The samples were loaded into agarose gel (1.2 %) 
coloured by GelRed (Biotium) and separated by 
electrophoresis. Tests were made in three repetition 
(A, B, C). As a positive sample was considered that 
one with two positive results from three repetition.
RESULTS AND DISCUSSION
For the presence of 6 virus pathogens were tested 
45 plants in 2010, from that 10 plants of variety 
Aurelius were tested in 2009 and 10 plants of the 
variety Neuburger were tested in 2005. The highest 
occurrence of virus pathogens was showed by 
Neuburger in the habitat Moravská Nová Ves, where 
leafroll virus GLRaV-1 was detected in 6 samples 
(100 %) and also GVA virus in 4 samples (66 %). It 
is possible to assume, that in that vineyard was 
planted infected propagation material. The mixed 
infection GVA and GLRaV-1 was found out in one 
plant Neuburger variety from Dolní Dunajovice and 
one plant of Neuburger from Dolní Kounice. Next 
most frequent occurrence was found out by GFkV 
at 3 plants of Neuburger in Dolní Kounice. Leafroll 
GLRaV-3 was found out at 2 plants, ArMV at one and 
GFLV at none plant. Altogether were 15 plants from 
45 tested infected at least by one virus (33.3 %), from 
270 made tests for viruses presence were 20 positive, 
e. g. 7.4 % (see Tab. II).
The phytoplasmas were detected only in one 
plant from 28 tested. In this plant was Potato stolbur 
phytoplasma confi rmed by other tests (Fig. 3). The 
plant was variety Aurelius from Dolní Dunajovice. 
It is clear, that phytoplasma is not the reason of 
abnormal dwarf growth of shoots. There were no 
typical visual symptoms of phytoplasma infection 
visible on these tested plants like sectorial yellowing 
of leaf or irregular ripening of shoots and gummy 
shoots (Červená et al., 2007).
The aff ected plants were slightly damaged by 
Colomerus vitis Pag. causing felting of leaves in 
habitats Dolní Dunajovice and Dolní Kounice in 
September. The reason of zig-zag growth of the 
branches could be in buds damage by mites during 
winter. The shoot from main bud sproots, but looses 
apical dominance, grows zig-zag and o en dies. The 
secondary branches from adventive buds become 
dominant later on and have little or none grapes 
(Walton et al., 2007).
The shoot base of plants of variety Aurelius in 
Čejkovice showed not only zig-zag growth, but also 
II: The results of virus and phytoplasmas detection in the plants of grapevine from 45 tested plants
Pathogen/variety
Habitats Number of 
infected plantsČejkovice D. Dunaj. D. Kounice M. N.Ves
ArMV
AUR 0 0 0 0
1
NEU 0 0 1 0
GFkV
AUR 0 0 0 0
3
NEU 0 0 3 0
GFLV
AUR 0 0 0 0
0
NEU 0 0 0 0
GLRaV-1
AUR 0 0 0 0
8
NEU 0 1 1 6
GLRaV-3
AUR 1 0 0 0
2
NEU 0 1 0 0
GVA
AUR 0 0 0 0
6
NEU 0 1 1 4
Phytoplasmas 
complex
AUR 0 1 0 0
1
NEU 0 0 0 0
Potato stolbur 
phytoplasma 
AUR 0 1 0 0
1
NEU 0 0 0 0
Consideration: ArMV Arabis mosaic virus; GFkV Grapevine fl eck virus; GFLV Grapevine fanleaf virus; GLRaV-1, 3 Grapevine leafroll 
assoc. virus 1 and 3; GVA Grapevine virus A; AUR Aurelius variety; NEU Neuburger
76 V. Holleinová, J. Čechová
blushing and bark-cracking, which can be caused 
not only by mites, but also by fungi disease which 
damage the xylem, or by nutrition lack.
CONCLUSION
Viral pathogens Arabis mosaic virus, Grapevine fl eck 
virus, Grapevine fanleaf virus, Grapevine leafroll assoc. 
viruses 1 and 3; Grapevine virus A and Potato stolbur 
phytoplasma were detected minimally in the tested 
plants Vitis vinifera L. damaged by dwarf shoots and 
clusters. This was not general occasion so-called 
short shoot syndrom. 
3: Picture of the agarose gel with samples positive to presence of Potato stolbur phytoplasma 
SUMMARY
The target of this work was verifi cation if the reason of grapevine shoot shortening is infection by virus 
pathogens and phytoplasmas. By serological method DAS-ELISA was checked the presence of 6 viruses in 
samples taken from 45 aff ected plants of grapevine on 4 habitats. The wood was tested during dormancy 
period, the leaves during vegetation. The presence of phytoplasmas was checked by method nested-PCR 
in the leaf samples. The highest occurrence of viruses was found out on habitat Moravská Nová Ves, where 
all taken samples were infected by Grapevine leafroll assoc. virus GLRaV-1 (100 %). In 66 % of the samples taken 
from that habitat were detected mixed infection of Grapevine virus A and GLRaV-1. These 2 pathogens but 
were not detected in samples from aff ected plants from the other habitats or very sporadically – just in 2 
plants. It is possible to assume, that in that vineyard was planted infected propagation material.
Another 4 virus pathogens were detected either sporadically (Grapevine fl eck virus, Grapevine leafroll assoc. 
virus GLRaV-3, Arabis mosaic virus) or not at all (Grapevine fanleaf virus). From 270 tests made to 6 viruses were 
only 20 positive, e. g. 7.4 %. It means that from 45 plants were 15 infected at least by 1 virus (33 %).
The phytoplasmas complex was tested in 28 plants. The result was positive only in 1 plant, by another test 
the Potato stolbur phytoplasma was confi rmed. The phytoplasmas do not cause that abnormal grapevine 
shoot shortening.
Acknowledgement
The research was done with fi nancial support of Ministry of Agriculture of the Czech Republic – 
program 3. c.
 The detection of viruses and phytoplasmas in dwarfed shoots of grapevine varieties Aurelius and Neuburger 77
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Address
Ing. Věra Holleinová, Ph.D., Mendeleum, Mendelova univerzita v Brně, Valtická 337, 691 44 Lednice, Česká 
republika, e-mail: holleinova@zf.mendelu.cz
78 


THE DETECTION OF VIRUSES AND PHYTOPLASMAS IN DWARFED SHOOTS OF GRAPEVINE VARIETIES AURELIUS AND NEUBURGER

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THE DETECTION OF VIRUSES AND PHYTOPLASMAS IN DWARFED SHOOTS OF GRAPEVINE VARIETIES AURELIUS AND NEUBURGER

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