PHYTOPLASMAS AND THEIR POTENTIAL VECTORS IN VINEYARDS OF INDIGENOUS
CROATIAN VARIETIES
Ivan Mikec1, Ivana Križanac1, Željko Budinščak1, Martina Šeruga Musić2, Mladen Krajačić2, Dijana
Škorić2*
Author’s addresses: 1Institute for Plant Protection in Agriculture and Forestry of the Republic of
Croatia, Svetošimunska 25, 10 000 Zagreb, Croatia; 2Department of Biology, Faculty of Science,
University of Zagreb, Marulićev trg 9a, 10 000 Zagreb, Croatia
*Correspondence to: dijana@botanic.hr, phone 00385 1 4843851, fax 00385 1 4844001.
Introduction
Grapevine yellows (GY) symptoms have been observed in most viticultural regions of Croatia. First
molecular evidence of aster-yellows (16SrI-B) and stolbur (16SrXII-A) phytoplasmas infecting Croatian
grapevines were presented by Šarić et al. (1997) and Škorić et al. (1998), respectively. Visual and
molecular surveys of indigenous grapevine cultivars from Croatian coastal vine-growing regions in the
period from 1998 to 2002 demonstrated only the presence of phytoplasmas belonging to the ribosomal
subgroup 16SrXII-A (Šeruga et al., 2002). In the years 2003 and 2004, the survey continued on a
larger scale encompassing indigenous cultivars from northern coastal, southern coastal and
continental viticultural areas of Croatia. In 2004, investigations of potential phytoplasma natural
vectors were started in three vineyards, one in each region.
Materials and methods
Leaves were collected from 20 symptomatic vines of 10 indigenous Croatian grapevine varieties from
different regions in September 2003 and 2004. In 2004, potential phytoplasma vectors were collected
by using yellow sticky traps and entomological net from three vineyards: Voloder (continental Croatia),
Novigrad and Pelješac (northern and southern coastal Croatia, respectively).
Total nucleic acids (TNA) were extracted following the procedure described by Šeruga et al. (2003).
Approximately 0.2 g of CaCl2-dried leaf midribs was used instead of the frozen/fresh material. TNA
from insects were extracted as described by Zhang et al. (1995).
Amplification of phytoplasma 16S rRNA gene was performed in a direct PCR using R16F1/R0
phytoplasma universal primer pair (Lee et al. 1995). First nested PCRs were performed by using
universal primer pairs R16F2n/R2 (Lee et al. 1993) and second nested PCRs were primed by groupspecific R16(I)F1/R1 and, in some cases, by R16(V)F1/R1 primers (Lee et al., 1994) as described by
Šeruga et al. (2000).
Amplicons from all nested PCR assays were subjected to digestion with restriction endonuclease
Tru9I (=MseI) for 16 h at 65°C. Obtained fragments were separated by electrophoresis through 5%
polyacrylamide gel, stained with ethidium-bromide and compared with RFLP patterns of standard
phytoplasma strains: SA-1, Stol, PTV (16SrXII-A), Hyd-B (16SrI-B), PPT (16SrI-C), EY-C (16SrV-A)
and KVI (16SrIII-B) (Bertaccini, 2003).
Results and discussion
GY symptoms were observed in all examined vineyards of indigenous Croatian varieties from different
viticultural areas (Table 1). In Plavac Mali and Babić milder GY symptoms (only leaf reddening) were
observed and phytoplasmas belonging to subgroups 16SrI-B and 16SrXII-A were detected,
respectively.
In direct PCR assays, no visible products were obtained. In nested PCR assays using generic primers,
only three samples (Malvasija and Škrlet) produced visible 1.2 kb products. Further PCR
amplifications using group–specific primer pair R16(I)F1/R1 yielded additional fifteen positive samples.
Plavec Žuti and one of the Plavac Mali samples tested negative. For samples showing R16(I)F1/R1/
Tru9I profiles characteristic of 16SrI-B subgroup, additional second nested PCR using R16(V)F1/R1
primers on R16F2n/R2 templates was done to exclude the possibility of mixed infections. The absence
of visible products primed by R16(V)F1/R1 indicates the presence of only 16SrI-B subgroup
phytoplasmas in these samples.
Six species of potential phytoplasma insect vectors were analyzed (Table 2). Insects collected in
vineyards not indicated in the literature as potential phytoplasma vectors, were not subjected to
laboratory analyses. In all vectors that resulted positive for phytoplasma presence, the agents
belonging to the subgroup 16SrXII-A were detected. The number of analyzed vectors was too small to
draw definite conclusions from these results. Nevertheless, Reptalus cuspidatus is abundantly present
and consistently harbors phytoplasmas from the subgroup 16SrXII-A suggesting its important role in
some of the pathosystems indigenous vines – vector – herbaceous hosts. Although our previous
research (not shown) preformed on a limited number of vineyard weeds indicates the involvement of
Taraxacum officinale, Stellaria media and Convovulus arvensis, the definite role of these, and possibly
other weeds for the molecular epidemiology of phytoplasmas in Croatian indigenous vines remains to
be determined.
Acknowledgements
The Croatian Ministry of Agriculture, Forestry and Water management financed this research.
References
Lee, I.M., Bertaccini, A., Vibio, M. & Gundersen, D.E., 1995. Detection of multiple phytoplasmas in perennial fruit
trees with decline symptoms in Italy. Phytopathology 85, 728-735.
Lee, I.M., Gundersen, D.E., Hammond, R.W. & Davis, R.E., 1994. Use of mycoplasmalike organism (MLO) groupspecific oligonucleotid primers for nested-PCR assays to detect mixed-MLO infections in a single host plant.
Phytopathology 84, 559-566.
Lee, I.M., Hammond, R.W., Davis, R.E. & Gundersen, D.E., 1993. Universal amplification and analysis of
pathogen 16S rDNA for classification and identification of mycoplasmalike organism. Phytopathology 83, 834-842.
Šarić, A., Škorić, D., Bertaccini, A., Vibio, M. & Murari E., 1997. Molecular detection of phytoplasmas infecting
grapevines in Slovenia and Croatia. Proc. 12th Meet. ICVG 1997, Lisbon, Portugal 77-78.
Šeruga, M., Ćurković Perica, M., Škorić, D., Kozina, B., Mirošević, N., Šarić, A., Bertaccini, A. & Krajačić, M.,
2000. Geographical distribution of bois noir phytoplasmas infecting grapevines in Croatia. Journal of
Phytopathology 148, 239-242.
Šeruga, M., Škorić, D., Kozina, B. & Krajačić, M., 2002. Phytoplasmas in Croatian indigenous grapevine cultivars.
Proc. 14th Int. Con. IOM 2002, Vienna, Austria.
Šeruga, M., Škorić, D., Botti, S., Paltrinieri, S., Juretić, N. & Bertaccini, A. F., 2003. Molecular characterization of a
phytoplasma from the aster yellows (16SrI) group naturally infecting Populus nigra L. 'Italica' trees in Croatia.
Forest Pathology 33, 113-125.
Škorić, D., Šarić, A., Vibio, M., Murari, E., Krajačić, M. & Bertaccini, A., 1998. Molecular identification and
seasonal monitoring of phytoplasmas infecting Croatian grapevines. Vitis 37, 171-175.
Zhang, Y.P., Uyemoto, J.K. & Kirkpatrick, B.C., 1995. A rapid, small scale procedure for extracting virus, viroid,
MLO and bacterial nucleic acid from plants for analysis by PCR. Phytopathology 85, 1205.
Bertaccini, A., 2003. URL: http://137.204.42.130/person/collection-september_2003.pdf. Date of last accession:
June 8, 2004.
Table 1. Phytoplasma identification in grapevine samples
Year
Region
Vineyard
Cultivar
Northern
coastal
Rovinj
Vrbnik
Pelješac
Čara
Lumbarda
Primošten
Zelina
Nespeš
Voloder
Malvasija
Žlahtina
Plavac Mali
Pošip
Grk
Babić
Kraljevina
Kraljevina
Moslavac
Kraljevina
Plavec Žuti
Plavac Mali
Plavac Mali
Pošip
Škrlet
Southern
coastal
2004
Continental
Križevci
2003
Southern
coastal
Continental
Pelješac
Čara
Voloder
Number of
samples
2
2
1
1
1
2
2
1
1
1
1
2
1
1
1
R16F2nR2/Tru9I
R16(I)F1/R1/Tru9I
16SrXII-A
16SrXII-A
not tested
16SrXII-A
16SrI-B
16SrXII-A
16SrXII-A
16SrXII-A
16SrXII-A
16SrXII-A
16SrI-B
16SrXII-A
16SrI-B
16SrI-B
not tested
Table 2. Phytoplasma identification in insect samples
Region
Vineyard
Northern coastal
Novigrad
Southern coastal
Pelješac
Continental
Voloder
Species
Reptalus cuspidatus
Scaphoideus titanus
Metcalfa pruinosa
Empoasca vitis
Arboridia dalmatina
Reptalus cuspidatus
Reptalus cuspidatus
Cicadella viridis
Reptalus panzeri
Hyalestes obsoletus
R16F2nR2/Tru9I
not tested
not tested
-
R16(I)F1/R1/Tru9I
16SrXII-A
-
16SrXII-A
16SrXII-A
-