66

ARTICLES B&E

BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/1

Keywords: Bulgarian grapevine viruses, grapevine genebank, certifi cation

IntroductionViticulture is an important industry in Bulgaria and grapevines are grown on approximately 129,580 ha. Virus diseases on grapevine have a signifi cant 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, classifi ed 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 certifi ed seed and planting material was adopted in

October 2000. A national system for production of certifi ed 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, Sofi a by DAS-ELISA technique for a few prevalent viruses. This paper describes in short the results obtained from virus testing of certifi ed grapevine planting material in Bulgaria for the last tree years.

Materials and MethodsPlant materialGrapevines 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 certifi ed 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 fl ack virus (GFkV), Grapevine virus A (GVA) and Grapevine virus B (GVB).

DAS - ELISA testsDAS-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. AtanassovAgroBioInstitute, Sofi a, 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 certifi ed 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 fl 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.

67BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/1

alkaline phosphatase conjugate produced in AgroBioInstitute, Sofi a, Bulgaria by d-r Jankulova and for GLRaV 3 identifi cation ELISA specifi c 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 identifi cation 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 A405 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 DiscussionThe 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 1Incidence of the tested viruses in grapevine varieties and rootstocks

Tested for: Number of tested vines % of positive vinesArMVGFLVGBLV

GLRaV 1GLRaV 2GLRaV 3GLRaV 7

GFkV GVA GVB

333105410549515657175659512304

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 fi 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 certifi cation 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 signifi cant 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).

ConclusionsThe control strategies of grapevine viruses are preventive. They are based on the identifi cation 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 certifi ed plants.

REFERENCES1. Abrscheva P., Rozenova L., Todorova M. (1994) Vitis,

33, 181-182. 2. Balo B., Veradi G.Y., Papp E., Muerdy, L.A., Poply

K.D. (1997) Structural and functional alterations in photosynthetic apparatus of virus-infected Chardonnay vines. Abstracts of 5th Int. Symposium on grapevine physiology, 25-30/05, Jerusalem.

3. Barba M., Cupidi A., Cassori L. (1993) Infl uence of virus and virus-like diseases of grapevine in shoot cultures. Extended abstracts 11th ICVG Meeting, Montreux, 43-44.

4. Credi R., Babini A.R. (1996) Adv. Hort. Sci., 10, 95-98.5. Gonzales E., Diaz M., Mosquera M.V. (1995) Vitis, 34,

243-244.

68 BIOTECHNOL. & BIOTECHNOL. EQ. 21/2007/1

6. Kovachevski I., Markov M., Yankulova M., Trifonov D., Stojanov D., Kacharmazov V. (1995) In: Virus and virus-like diseases of crop plants. PSSA, 277-284.

7. Legin R., Bass P., Etienne, L., Fucks M. (1993) Vitis, 32, 103-110.

8. Martelli G.P. (2000) Grapevine virology high-lights 1997-1999. Extended abstracts 13th ICVG Conf., 1-5, Adelaide, Australia.

9. Martelli G.P. (2002) Inforamatore fi topatogico, 4, 18-27. 10. Walter B. (1997) In: Les Colloques, INRA Iditions 86, p.

225.