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Informatore Agrario 43(49): 69-75. Walker J.T.S., Charles J.G., Froud K.J., Connolly P., 2004. Leafroll virus in vineyards: modelling the spread and eco-.
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Journal of Plant Pathology (2009), 91 (3), 741-744

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SHORT COMMUNICATION MONITORING THE SPREAD OF VIRUSES AFTER VINEYARD REPLANTING WITH HEAT-TREATED CLONES OF VITIS VINIFERA ‘NEBBIOLO’ I. Gribaudo1, G. Gambino1, S. Bertin2, D. Bosco2, A. Cotroneo3 and F. Mannini1 1 Istituto

Virologia Vegetale del CNR, Unità di Grugliasco, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy di Valorizzazione e Protezione delle Risorse Agroforestali, Università degli Studi di Torino, settore Entomologia e Zoologia Applicate all’Ambiente ‘Carlo Vidano’, Via L. da Vinci 44, 10095 Grugliasco (TO), Italy 3 Regione Piemonte, Settore Fitosanitario Regionale, Via Livorno 60, 10144 Torino, Italy 2 Dipartimento

SUMMARY

Two vineyards planted in the Langhe (Piedmont, Italy) with clonal vines of Vitis vinifera cv. Nebbiolo originating from heat-treated mother plants, were monitored for the occurrence and spread of viral infections. More than ten years after the establishment of one of the vineyards, where rows of vines infected with Grapevine virus A (GVA) and Grapevine leafroll-associated virus 1 (GLRaV1) or GVA and Grapevine leafroll-associated virus 3 (GLRaV-3) were interplanted with rows of healthy plants, viruses had spread to 18.5% of the healthy vines. Spreading was slow and limited to small groups of vines, the size of which increased slowly, thus providing indirect evidence of transmission by vectors. Mealybugs collected in the vineyard were classified as Heliococcus bohemicus. RT-PCR analysis of mealybugs revealed the presence of at least one virus in more than 45% of the tested insect batches, indicating that H. bohemicus can acquire viruses during feeding on infected plants, thus acting as a potential vector. No natural spread of Grapevine fanleaf virus (GFLV) was detected in a second vineyard which had been fumigated before re-planting, because of the heavy incidence of fanleaf disease in the preceding uprooted vineyard, and where Xiphinema index, the GFLV vector, was found only occasionally. Key words: grapevine viruses, vector transmission, mealybugs, nematodes, Heliococcus bohemicus, epidemiology.

Virus diseases are still a major threat to grapevines (Vitis spp.). Decades if not centuries of cultivation in the same areas, due to the favourable climate and soil, and often to the lack of alternative crops, have led to a stable presence of grape pests and pathogens, including viruses. Planting vineyards with virus-free plants (originally healthy or sanitized) is the only preventive measure to lower the incidence and spread of viral infections. Corresponding author: I. Gribaudo Fax: +39.011.6708658 E-mail: [email protected]

However, when a vineyard is planted or re-planted with certified virus-free material, viruses that may be present in nearby vineyards can be moved to the new stand by vectors. Several species of mealybugs (Pseudococcidae) and soft scale insects (Coccidae) are known to transmit viruses involved in the aetiology of leafroll and rugose wood, i.e. Grapevine leafroll associated virus 1 and 3 (GLRaV-1 and GLRaV-3) and Grapevine virus A (GVA) (Cabaleiro and Segura, 1997; Engelbrecht and Kasdorf, 1990; Fortusini et al., 1997; La Notte et al., 1997; Golino et al., 2002), whereas the nematode Xiphinema index is the vector of Grapevine fanleaf virus (GFLV), the causal agent of fanleaf disease (Andret-Link et al., 2004). Early infections caused by vector-mediated transmission of viruses can reduce the preventative advantage of planting healthy vines, thus lowering the benefit of their use. Therefore, a better knowledge of how viruses spread in vineyards established with healthy plants will be useful, also from an economic point of view. Since virus epidemics characterized by different infection rates and rapidity of virus spread have been reported from different countries (Kasdorf and Engelbrecht, 1990; Belli et al. 1993; Cabaleiro and Segura, 1997; Habili and Nutter, 1997; Walker et al., 2004; Pietersen, 2006), the aim of this investigation was to monitor the natural re-infection rates of healthy clonal vines under Italian conditions. The study was carried out in two experimental plots henceforth called “Neive” and “Barbaresco,” located in the Langhe, an area of intensive viticulture in northwestern Italy. Both plots were previous vineyards and were re-planted with clonal vines of cv. Nebbiolo (Vitis vinifera L.), grafted onto certified rootstocks which, before use, had been randomly tested by ELISA for the absence of viruses. Vines were vertically trained and cane pruned (Guyot system), with a spacing of 2.7 m between rows and 1 m along the row. The Neive vineyard, established in November 1992, consists of 19 rows of 45 plants each. Five non-adjacent rows were planted with infected vines (5th, 11th and 17th row, GVA+ GLRaV-3; 7th and 13th row, GVA+GLRaV1), while all the other rows were planted with vines of the same clones free from the mentioned viruses. Moth-

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er plants of the clones were originally doubly infected by GVA in association with GLRaV-1 or GLRaV-3, which were eliminated by in vivo heat-therapy, as ascertained by repeated ELISA testing. The experimental plot was surrounded by commercial vineyards of unknown virological status. The Barbaresco vineyard was planted with vines propagated from heat-treated GFLV-free mother plants. Because of the heavy incidence of fanleaf disease in the preceding vineyard and in the surrounding ones, the plot had been fumigated in 1993, one year prior to replanting with healthy vines. Analyses for the presence of viruses and their potential vectors were done in 2005, 2006 and 2007, more than 10 years after plantings. Serological assays were carried out as previously described (Gambino et al., 2006) using cortical scrapings from mature canes collected in winter. The sanitary status of the Neive vineyard was investigated also in 2004, sampling all the orig-

Journal of Plant Pathology (2009), 91 (3), 741-744

inally healthy plants of the 4th, 6th and 12th rows, whereas in the Barbaresco vineyard, 25 to 56 vines scattered throughout the plot were assayed, depending on the year. Polyclonal antisera and monoclonal antibodies used for testing were from Agritest (Italy). In 2007 the vineyards were inspected in July, August and September for potential insect vectors. Mealybugs were collected from infected vines (5th, 7th, 11th, 13th and 17th rows) at Neive, and identified based on the morphology of adult females. Since mealybug nymphs are not readily distinguishable and Planococcus citri Risso and P. ficus Signoret are often found in vineyards, the mitochondrial cytochrome oxidase I (COI) gene was amplified using species-specific primers (Saccaggi et al., 2008). Amplification products obtained from nymphs were sequenced and compared with those of Planococcus species available in GenBank. Mealybug nymphs were tested for GLRaV-1, GLRaV3 and GVA by RT-PCR. RNA was extracted from nine

Fig. 1. Evolution of virus infections in the Neive vineyard as detected by ELISA.

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groups of five insects each using TRIzol (Invitrogen Life Technologies, USA). First-strand cDNA was synthesized from total RNA as previously described (Gambino et al., 2006). Viruses were detected with conventional (Gambino et al., 2006) and real time PCR with specific primers designed on the viral coat protein sequences (GVA fw GTGGTGTCGGAGCTGGTC and GVA rev ACCTAGTCATCTTGGTGGCTAAC; GLRaV-1 fw CGTTTGAAAATCCTATGCGTCAG and GLRaV-1 rev GCAACTTTCTCGTTCGGCTTC; GLRaV-3 fw TTCGAGAAAGATCCAGACAAGTTC and GLRaV-3 rev ATAACCTTCTTACACAGCTCCATC). Nematode surveys were carried out in the Barbaresco vineyard from 2005 to 2007, assessing population density from soil samples of 500 cm3, collected at a depth of 30-50 cm in late spring and autumn, in six spots inside the fumigated vineyard and, as a control, in four spots in the bordering commercial vineyards. ELISA assays of samples from the Barbaresco vineyard were always negative for GFLV. In the Neive vineyard, the overall infection rate by GLRaV-1, GLRaV-3 and GVA, as ascertained by ELISA, rose from 7.4% in 2004 to 18.5% in 2007 (Fig. 1). Virus spread was slow and limited to small groups of vines, the size of which increased slowly, thus providing indirect evidence of transmission by slow moving vectors. Mealybugs collected in summer were examined microscopically and classified as Heliococcus bohemicus Sulc (family Pseudococcidae). COI sequences obtained from nymphs did not match those of P. citri or P. ficus, indicating that they actually belong to a different species (COI sequence of H. bohemicus is not available in GenBank). H. bohemicus has previously been reported as vector of GLRaV-1 and GLRaV-3 (Sforza et al., 2003) and of GLRaV-3 and GVA (Zorloni et al., 2006). In the Neive vineyard, H. bohemicus population increased during the season with a peak in September-October with nymphs of the second generation which overwinter under the grapevine bark. RT-PCR analyses showed the presence of the three viruses (GVA, GLRaV1 and GLRaV-3) in the individuals collected in this vine-

Gribaudo et al. 70,0 60,0 50,0 40,0

adjacent vineyards

30,0

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20,0 10,0 0,0 june june june 05 06 07

sept sept sept 05 06 07

Fig. 2. Total number of plant parasitic nematodes found in the soil of the Barbaresco experimental vineyard and of adjacent vineyards. For each date and zone, data represent an average of 3 to 7 soil samples (500 cm3 each).

yard (Tab. 1). Mealybugs sampled from another cv. Nebbiolo vineyard in the same area were again H. bohemicus, and proved to be RT-PCR positive for GLRaV-1 and GLRaV-3 (five insect batches of 13 tested). These data are taken as confirmatory evidence that H. bohemicus can acquire the viruses in question during feeding on infected plants, thus representing their potential vector. The rate of GLRaV-3 spread reported by others (Pietersen, 2006; Cabaleiro and Segura, 2006; Cabaleiro et al., 2008) is much faster than what we registered. Possible explanations are: (i) different mealybug species [H. bohemicus in Piedmont (Italy) vs P. citri in Galicia (Spain)]; (ii) different mealybug population levels; (iii) different size of infection foci related to the age of the vineyard (Cabaleiro and Segura, 2003). Nematode analyses performed on soil samples from the Barbaresco vineyard showed the presence of Helicotylenchus spp., Xiphinema pachtaicum (Fig. 2) and saprophytic species, the latter being the most common. X. index specimens were found only once in the Barbaresco vineyard (September 2006) and never outside of it, thus confirming the low frequency of this nema-

Table 1. Real time RT-PCR detection of GVA, GLRaV-1 and GLRaV-3 in Heliococcus bohemicus nymphs sampled in 2007 (Neive vineyard).

Collection time July July July August August August September September September

N° of insects/batch 5 5 5 5 5 5 5 5 5

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GLRaV-1

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tode in the Piedmontese vineyards (Tacconi and Mancini, 1987; A. Cotroneo, unpublished information). This is in line with the notion that the widespread occurence of fanleaf disease in old Piedmontese vineyards depended primarily on the use of infected propagating material, rather than on active dissemination by nematodes. The present work, started in 2005, allowed to monitor the spread of air-borne viruses in a vineyard (Neive), exposed to fairly high risks of reinfection. More than ten years after re-planting, the spread of GVA, GLRaV1 and GLRaV-3 was still limited (less than 20% of newly infected plants) notwithstanding conditions (infected vines interplanted with sanitized vines) that should have favoured natural virus dissemination in the presence of mealybug vectors. No natural spread of fanleaf disease has yet been detected in the Barbaresco vineyard where the nematode X. index (vector of GFLV) was found only occasionally. All this may be taken as an indication that, under the conditions in which the experiment was conducted, the use of healthy material can guarantee the maintenance of an acceptable sanitary status long enough to be economically profitable. Further monitoring in the years to come will provide an ultimate answer. ACKNOWLEDGEMENTS

Research funded by the Regione Piemonte. The authors thankfully acknowledge the help of D. Cuozzo, N. Argamante, D. Dellavalle, and of Maggie Sherriffs for language revision. REFERENCES Andret-Link P., Laporte C., Valat L., Ritzenthaler C., Demangeat G., Vigne E., Laval V., Pfeiffer P., Stussi-Garaud C., Fuchs M., 2004. Grapevine fanleaf virus: still a major threat to the grapevine industry. Journal of Plant Pathology 86: 183-195. Belli G., Fortusini A., Prati S., 1993. Natural spread of grapevine leafroll disease in a vineyard of Northern Italy. Extended Abstracts 11th Meeting of ICVG, Montreaux 1993: 110. Cabaleiro C., Segura A., 1997. Field transmission of Grapevine leafroll associated virus 3 (GLRaV-3) by the mealybug Planococcus citri. Plant Disease 81: 283-287. Cabaleiro C., Segura A., 2003. Monitoring the field spread of Grapevine leafroll-associated virus 3 for 12 years. Extended Abstracts 14th Meeting of ICVG, Locorotondo 2003: 216-217. Cabaleiro C., Segura A., 2006. Temporal analysis of Grapevine leafroll associated virus 3 epidemics. European Journal of Plant Pathology 114: 441-446.

Received March 26, 2009 Accepted July 14, 2009

Journal of Plant Pathology (2009), 91 (3), 741-744 Cabaleiro C., Couceiro C., Pereira S., Cid M., Barrasa M., Segura A., 2008. Spatial analysis of epidemics of Grapevine leafroll associated virus-3. European Journal of Plant Pathology 121: 121-130. Engelbrecht D.J., Kasdorf G.G.F., 1990. Transmission of grapevine leafroll disease and associated closterovirus by the vine mealybug Planococcus ficus. Phytophylactica 22: 341-346. Fortusini A., Scattini G., Prati S., Cinquanta S., Belli G., 1997. Transmission of Grapevine leafroll virus 1 (GLRaV1) and Grapevine virus A (GVA) by scale insects. Extended Abstracts 12th Meeting of ICVG, Lisbon 1997: 121-122. Gambino G., Bondaz J., Gribaudo I., 2006. Detection and elimination of viruses in callus, somatic embryos and regenerated plantlets of grapevine. European Journal of Plant Pathology 114: 397-404. Golino D.A., Sim S.T., Gill R., Rowhani A., 2002. California mealybugs can spread grapevine leafroll disease. California Agriculture 56 (6): 196-201. Habili N., Nutter F.W.Jr., 1997. Temporal and spatial analysis of Grapevine leafroll-associated virus 3 in Pinot noir grapevines in Australia. Plant Disease 81: 625-628. La Notte P., Buzkan N., Choueiri E., Minafra A., Martelli G.P., 1997. Acquisition and transmission of Grapevine virus A by the mealybug Pseudococcus longispinus. Journal of Plant Pathology 78: 79-85. Kasdorf G.G.F., Engelbrecht D.J., 1990. Field spread of corky bark, fleck, leafroll and Shiraz decline diseases and associated viruses in South African grapevines. Phytophylactica 22: 347-354. Pietersen G., 2006. Spatio-temporal distribution dynamics of grapevine leafroll disease in Western Cape vineyards. Extended Abstracts 15th Meeting of ICVG, Stellenbosch 2006: 126-127. Saccaggi D.L., Krüger K., Pietersen G., 2008. A multiplex PCR assay for the simultaneous identification of three mealybug species (Hemiptera: Pseudococcidae). Bulletin of Entomological Research 98: 27-33. Sforza R., Boudon-Padieu E., Greif C., 2003. New mealybug species vectoring Grapevine leafroll-associated viruses-1 and -3 (GLRaV-1 and -3). European Journal of Plant Pathology 109: 975-981. Tacconi R., Mancini G., 1987. I nematodi associati alla vite. Informatore Agrario 43(49): 69-75. Walker J.T.S., Charles J.G., Froud K.J., Connolly P., 2004. Leafroll virus in vineyards: modelling the spread and economic impact. HortResearch Client Report 12795, The Horticulture and Food Research Institute of New Zealand Ltd (http://www.nzwine.com/reports/). Zorloni A., Prati S., Bianco P.A., Belli G., 2006. Transmission of Grapevine virus A and Grapevine leafroll-associated virus 3 by Heliococcus bohemicus. Journal of Plant Pathology 88: 325-328.