66

ARTICLES B&E
BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/1
Keywords: Bulgarian grapevine viruses, grapevine genebank,
certication
Introduction
Viticulture is an important industry in Bulgaria and grapevines
are grown on approximately 129,580 ha. Virus diseases on
grapevine have a signicant economic impact on plant growth,
yield and fruit quality, as they can affect the graft compatibility,
rooting capacity, winter hardiness, longevity of the vines and
to lead to plant mortality (10). More than 53 viruses have been
detected in Vitis (8, 9) and at least 8 viruses of them have been
found in Bulgaria (6).
All known grapevine viruses are graft-transmissible.
They are spread long distances through infected propagating
material (buds/cuttings), or by the use of rooting cuttings
from infected canes. Additionally, several grapevine viruses,
classied as nepoviruses, including Grapevine fanleaf virus
(GFLV), Arabis mosaic virus (ArMV), Tomato black ring
virus (ToBRV), Tomato ringspot virus (ToRSV) and Tobacco
ringspot virus (TRSV) are transmitted in short distances by
certain species of nematodes.
The control of grapevine viruses is dependent upon the
effectiveness of clean stock programs, where virus-free
propagation material is used in nurseries and vineyards.
Addressing the problem of the sanitary improvement of
vegetative propagated crops, the EEC issued and acted
Directive 68/193, complemented by Directive 00/11 containing
provisions for the production of grapevine material free from a
couple of virus diseases (fanleaf and leafroll). In Bulgaria, as a
pre-accession country this Directive was applied promptly and
a law concerning the production of virus-free and virus-
tested certied seed and planting material was adopted in
October 2000. A national system for production of certied
vine growing material was established by the Ministry of
Agriculture and Forestry with the help of FAMAD project to
the German Society of Technical Assistance (GTZ).
Since 2000 the propagated grape materials are checked in
AgroBioInstitute, Soa by DAS-ELISA technique for a few
prevalent viruses. This paper describes in short the results
obtained from virus testing of certied grapevine planting
material in Bulgaria for the last tree years.
Materials and Methods
Plant material
Grapevines were selected by their virology status from several
vine-growing areas of Bulgaria in the period of 2004-2006.
The surveys were based on the necessity to check and to select
healthy grapevines with the aim to produce certied material by
plant propagation (in situ or/in in vitro). Totally 1295 samples
from V. vinifera, 77 rootstock samples and 69 samples from V.
vinifera ssp. sylvestris were tested for some of the following
viruses: Arabis mosaic virus (ArMV), Grapevine Bulgarian
latent virus (GBLV), Grapevine fanleaf virus (GFLV),
Grapevine leafroll associated viruses 1, 2, 3 and 7 (GLRaV 1,
2, 3 and 7), Grapevine ack virus (GFkV), Grapevine virus A
(GVA) and Grapevine virus B (GVB).
DAS - ELISA tests
DAS-ELISA detection of ArMV, GFLV, GFkV, GVA, and
GLRaV 1 was performed with commercially available
polyclonal antibodies, immunoglobulin G (IgG), and alkaline
phosphatase-conjugated IgG (Bioreba, Reinach, Switzerland),
according to manufactures instructions. For GBLV, DAS-
ELISA tests were conducted with polyclonal antibodies and
VIRUS TESTING OF CERTIFIED GRAPEVINE PLANTING MATERIAL IN
BULGARIA
I. Kamenova, I. Tsvetkov, A. Atanassov
AgroBioInstitute, Soa, Bulgaria
Correspondence to: Ivanka Kamenova
Email: vkamenova@abi.bg
ABSTRACT
In accordance with the European Legislation and the recently developed national system for production of certied vine growing
material, 1295 samples from grapevine varieties and rootstocks were checked for their virus status in the period of 2004-
2006. The results obtained showed that total of 30.50% of the ELISA tested Vitis vinifera vines and rootstocks were infected
by one (86.08%) or more viruses (13.92). The most widespread virus was Grapevine ack virus (GFkV) (23.3%), followed
by Grapevine fanleaf virus (GFLV) (5.05%) and Grapevine leafroll associated viruses 1 and 3 (GLRaV 1 and 3) (3.34% and
2.78%), respectively. Grapevine Bulgarian latent virus (GBLV) (0.37%) was scarcely represented, while Arabis mosaic virus
(ArMV), Grapevine leafroll associated viruses 2 and 7 (GLRaV 2 and 7), Grapevine virus A (GVA) and Grapevine virus B (GVB)
were completely absent. The mixed infection showed eight virus combinations. The associations of GFkV + GLRaV 1 (8.3%) and
GFLV + GLRaV 3 (8.3%) were the most widespread ones.
All tested for ArMV, GFLV and GBLV wild vines (Vitis vinifera ssp. sylvestris) were virus free.
67 BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/1
alkaline phosphatase conjugate produced in AgroBioInstitute,
Soa, Bulgaria by d-r J ankulova and for GLRaV 3 identication
ELISA specic kit (Agritest, Italy) was used.
The following tissue samples were utilized: (i) several young
fully developed leaves from each plant for GFLV, ArMV and
GBLV identication during the spring and (ii) several mature
leaves per plant during the fall season for DAS-ELISA tests of
GLRaV 1, 2, 3 and 7, GFkV, GVA and GVB.
Controls of sample buffer only, uninfected and infected
leaves were included in all assays. Two wells were used for
each sample with the mean becoming the A
405 nm
reading for the
sample. Absorbance values were read after 2 h of incubation.
Samples were considered positive when the absorbance value
was at least three times greater than the absorbance value of
the healthy control.
Results and Discussion
The sanitary status of Bulgarian viticulture is little known
as few published reports are available. Given the paucity of the
information concerning the incidence of virus infection in vine
the present investigation was initiated. The results obtained are
summarized (Table 1).
TABLE 1
Incidence of the tested viruses in grapevine varieties and
rootstocks
Tested for: Number of tested vines % of positive vines
ArMV
GFLV
GBLV
GLRaV 1
GLRaV 2
GLRaV 3
GLRaV 7
GFkV
GVA
GVB
333
1054
1054
951
565
717
565
951
230
4
0.0%
5.05%
0.37%
2.78%
0.0%
3.34 %
0.0%
23.36%
0.0%
0.0%
A total of 30.50% of the ELISA tested Vitis vinifera vines
and rootstocks (395 out of 1295) were infected by one (86.08%)
or more viruses (13.92). GFkV was the most widespread virus
(23.3%), followed by GFLV (5.05%), GLRaV 3 (3.34%) and
GLRaV 1 (2.78%). GBLV (0.37%) was scarcely represented,
while ArMV, GLRaV 2 and 7, GVA and GVB were completely
absent.
The frequency of viruses detected in mixed infection could
be summarized by the following: GFkV +GLRaV 1 (8.3%)
=GFLV +GLRaV 3 (8.3%) >GFkV +GLRaV 3 (6.7%) >
GFkV +GFLV (4.2%) >GFLV +GLRaV 3 (3.6%) >GFLV +
GLRaV 1 (3%) >GLRaV 1 +GLRaV 3 (0.5). Triple infection
of GFLV +GLRaV 1 +GLRaV 3 viruses was found at a rate
of 0. 4%.
Compared to the tested grapevine varieties markedly well
was the sanitary condition of the tested rootstocks, since all
tested 77 rootstocks were virus free.
The tested 68 wild vines (Vitis vinifera ssp. sylvestris)
collected from six different regions of the country with the
aim of lling in the existing in AgroBioInstitute grapevine
genebank reacted negative for ArMV, GFLV and GBLV.
A survey in Bulgaria of grapevine varieties made in
the period of 1999-2003 in AgroBioInstitute has shown an
infection of 17.16% % of GFkV (Yankulova, unpublished
results). Unlike our results the same author has found GBLV
to be the second most distributed virus in the tested plants
(13.92%), followed by GFLV (9.88%).
According to the Bulgarian certication scheme, ArMV,
GFLV, GLRaV 1 and 3 and GFkV should be eliminated from
propagating vine material. In that aspect the results obtained
showed that the health conditions of the tested propagating
material especially in the case of GFkV and GFLV are
worrying. Both viruses are associated with their adverse
effects on infected plants. A dramatic yield reduction (60%)
in Chardonnay vines infected by GFLV in Hungary has been
reported (2) and signicant reduction in pruning weight, yield
and bunch size has been recorded in clonal plants of three V.
vinifera cvs. graft-inoculated with different strains of GFLV
and ArMV (7). Infected with GFLV vines propagated by
in vitro culture have shown very poor growth and rooting
capacity (1, 3, 5) and high decrease of the cutting production
from rootstocks mother-vine infected with GFLV have been
noted (4).
Conclusions
The control strategies of grapevine viruses are preventive.
They are based on the identication and destruction of infected
material to reduce disease incidence and minimize economic
damage. The results of our investigation highlight the need of
further assessment of grapevine virus status in Bulgaria in the
production of basic certied plants.
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