survey of fig viruses in the canary islands - SIPaV

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+. Brevera. Tenerife (ICIA) Tenerife Valle Guerra. +. -. +. +. -. +. Bicariña. Tenerife (ICIA) Tenerife Valle Guerra. -. -. -. +. -. +. Cuello Dama. Tenerife (ICIA) Badajoz.
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Journal of Plant Pathology (2011), 93 (3), 737-739

Edizioni ETS Pisa, 2011

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SHORT COMMUNICATION SURVEY OF FIG VIRUSES IN THE CANARY ISLANDS T. Elbeaino1, Á.M. González Rodríguez2, M.J. Grajal-Martín2 and M. Digiaro1 1Istituto

Agronomico Mediterraneo, Via Ceglie 9, 70010 Valenzano, Bari, Italy de Fruticultura Tropical, Instituto Canario de Investigaciones Agrarias, Apdo. 60, 38200 La Laguna Tenerife, España

2Departamento

SUMMARY

A survey for viruses was carried out in the main figgrowing areas of the Canary Islands in 2010. A total of 30 samples were collected and checked by RT-PCR for the presence of Fig mosaic virus (FMV), Fig leaf mottleassociated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FLMaV-2), Fig mild mottling-associated virus (FMMaV) and Fig latent virus 1 (FLV-1) using virusspecific primers. Approximately 90% of the trees were infected by at least one virus, with mixed infections in ca. 67% of the samples. FLMaV-1 and FMV were the prevailing viruses as they were found in 53.3% and 50% of the samples, respectively. Equally significant was the infection rate of the other three viruses investigated. Mosaic symptoms were consistently observed in most FMV-infected fig trees (43%). Few trees with mosaic symptoms (17%), however, were RT-PCR negative for FMV. These trees were mix-infected with most of the other tested viruses. Key words: Ficus carica, RT-PCR, fig mosaic, detection.

Fig (Ficus carica), a traditional fruit tree of the Mediterranean area, is also grown in the Canary Islands, where fruits are used for fresh and dried consumption. Currently, most of the figs in these islands grow as isolated plants in private gardens for family use. These trees show a wide range of discolorations (chlorotic mottling and blotching, banding, clearing and feathering of the veins, chlorotic ringspots and line patterns) and malformations of the leaves, recalling the symptoms of fig mosaic disease (FMD) (Martelli, 2011). FMD is a graft-transmissible disease (Condit and Horne, 1933) vectored by the eriophyid mite Aceria ficus (Flock and Wallace, 1955; Slykhuis, 1973), to which membrane-bound, round to ovoid bodies 90-200 nm in diameter and/or elongated bacilliform structures up to Corresponding author: T. Elbeaino Fax: +39-0804606275 E-mail: [email protected]

or above 1 µm in length, referred to by Bradfute et al. (1970) as double-membrane bodies (DMBs), are consistently associated. DMB were recently shown to be particles of Fig mosaic virus (FMV), a putative species of the family Bunyaviridae (Elbeaino et al., 2009). However, symptomatic figs of different geographical origin frequently contain also viruses of the family Closteroviridae, i.e. Fig leaf mottle-associated virus 1 (FLMaV-1), Fig leaf mottle-associated virus 2 (FLMaV2) and Fig mild mottle associated virus (FMMaV) (Elbeaino et al., 2006, 2007, 2010), and Fig latent virus 1 (FLV-1), another filamentous virus of the family Flexiviridae, which occurs also in symptomless trees and seedlings (Gattoni et al., 2009). To gather information on the virus population present in FMD-affected trees of the Canary Islands, a preliminary survey was carried out in the autumn of 2009 in the main fig-growing areas of El Hierro and Gran Canaria and in a germplasm collection of the Instituto Canario de Investigaciones Agraria (ICIA) at Tenerife (Table 1). A total of 30 symptomatic and symptomless fig trees of different varieties were sampled at El Hierro (9), Gran Canaria (8) and Tenerife (13). Samples consisting of leaves and cuttings about 30 cm in length were collected from one- to two-year-old shoots from the quadrant of the tree canopy and stored in plastic bags at 4°C until use for laboratory assays. Total nucleic acids (TNAs) were extracted from 100 mg of leaf vein tissues or cortical scrapings macerated in 1 ml grinding buffer (4.0 M guanidine thio-cyanate, 0.2 M NaOAc pH 5.2, 25 mM EDTA, 1.0 M KOAc and 2.5% w/v polyvinylpyrrolidone 40), with a silica capture procedure (Foissac et al., 2001), and stored at -20°C until used. Glasshouse-grown 2-year-old healthy fig seedlings from Apulia (southern Italy) were used as negative controls. Eight to 10 µl of TNA extract were mixed with 0.5 µl of random hexamer primers (Boehringer, Germany) (0.5 µg/µl), denatured at 95°C for 5 min and quickly chilled in ice. Reverse transcription reaction was done for 1 h at 39°C by adding 4 µl buffer 5X (50 mM trisHCl pH 8.3, 75 mM KCl, 3 mM MgCl2), 2 µl of 10 mM DTT, 0.5 µl of 10 mM dNTPs, and 200 units of Moloney murine leukaemia virus (M-MLV) reverse tran-

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Fig viruses in the Canary Islands

Journal of Plant Pathology (2011), 93 (3), 737-739

Table 1. Viruses detected by RT-PCR on fig trees in the Canary Islands. Cultivar

Island

Locality

FLMaV-1

FLMaV-2

FMMaV

FMV

FLV-1

Mosaic-like symptoms

Gomera

El Hierro

El Julán

-

-

+

+

-

+

Blanca

El Hierro

Sabinosa

+

-

+

-

-

-

Bicariña

El Hierro

Isora

+

-

-

-

+

-

Brevera Bicariña

El Hierro

El Pinar

+

+

+

-

+

+

Tipo Bicariña

El Hierro

El Pinar

+

-

+

-

+

+

Negra

El Hierro

El Tejal

+

-

-

-

+

-

Cotia

El Hierro

Isora

+

+

+

-

+

+

Brevera cotia

El Hierro

Isora

-

-

-

-

-

-

Morada

El Hierro

Tajase

+

-

+

-

+

+

Blanca de invierno

Gran Canaria

Guía

-

-

-

-

-

-

da Libra

Gran Canaria

Artenara

-

-

-

-

+

-

Brigazote

Gran Canaria

Artenara

-

-

-

+

-

-

Bicariña negra

Gran Canaria

Artenara

-

-

-

-

-

-

Bicariña amarilla

Gran Canaria

Artenara

-

-

-

+

-

+

Higuera canaria

Gran Canaria

Artenara

+

-

-

+

-

-

Negra

Gran Canaria

Artenara

+

+

-

+

-

+

Cota amarilla

Gran Canaria

Artenara

+

-

+

+

-

+

Mulata

Tenerife (ICIA)

Tenerife Fasnia

-

-

+

-

-

-

Blanca

Tenerife (ICIA)

Tenerife Fasnia

+

-

-

-

-

-

da Libra

Tenerife (ICIA)

Tenerife Fasnia

-

+

+

-

+

+

Blanca escalona

Tenerife (ICIA)

Gran Canaria La Granja

+

+

-

+

-

+

Gomera

Tenerife (ICIA)

Tenerife Valle Guerra

-

-

-

+

-

+

Brevera

Tenerife (ICIA)

Tenerife Valle Guerra

+

-

+

+

-

+

Bicariña

Tenerife (ICIA)

Tenerife Valle Guerra

-

-

-

+

-

+

Cuello Dama

Tenerife (ICIA)

Badajoz

+

+

+

+

-

+

Nazaret

Tenerife (ICIA)

Badajoz

-

+

-

-

-

-

San Antonio

Tenerife (ICIA)

Badajoz

-

-

+

+

-

+

Tiberio

Tenerife (ICIA)

Badajoz

+

+

+

+

+

+

Albar

Tenerife (ICIA)

Badajoz

+

+

-

+

+

+

Negra comun

Tenerife (ICIA)

Badajoz

Infected

+

+

+

(No.)

16

10

13

15

10

18

(%)

53.3

33.3

43.3

50.0

33.3

60.0

scriptase (Bethesda Research Laboratories, USA) in 20 µl as a final volume. For RT-PCR detection of five fig viruses (FMV, FLMaV-1, FLMaV-2, FMMaV, FLV-1) the virus-specific primers and the cycling conditions reported by El-

beaino et al. (2006, 2007, 2009, 2010) and Gattoni et al. (2009) were used. 2.5 µl cDNA mixture was submitted to amplification by adding 2.5 µl of 10X Taq polymerase buffer (Promega, USA), 1.5 mM final concentration of MgCl2, 0.5 µl of 10 mM dNTPs, 0.5 µl of 10 µM

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Fig. 1. RT-PCR amplifications obtained using specific primers for the detection of: (a) FLMaV-1, lanes, 2, 3, 5, 6, 8, 9; (b) FLMaV2, lanes 4, 5, 6, 8; (c) FMMaV, lanes 2, 3, 4, 5, 6, 8; (d) FMV, Lanes 2, 3, 4, 6, 7 and (e) FLV-1, lanes 2, 3, 4, 5, 7. Positive controls, lane 10 (a,b,c,d,e). Negative controls, lane 7 (a) and lane 9 (b,c,d,e). Lane 1, DNA size marker (Fermentas, Italy). All infected samples yielded an amplified product of the expected size whereas no amplifications were obtained from samples that did not contain the specific virus.

sense and antisense specific primers, and 0.2 µl of Taq polymerase (5 unit/µl) in a final volume of 25 µl. As shown in Table 1, of the 30 samples tested by RTPCR, 27 (90%) were infected by at least one of the five tested viruses, while the incidence of mixed infections was over 60% (19 of 30 trees). FLMaV-1 and FMV were the prevailing viruses with infection rates of 53.3% and 50%, respectively, followed by FMMaV (43.3%), FLV-1 (33.3%) and FLMaV-2 (33.3%) (Fig. 1; Table 1). Of the 12 trees that did not show apparent FMD-like symptoms at the time of the survey 8 were FMV-negative, whereas 15 (88%) of the 17 symptomatic plants were FMV-positive, thus confirming the high level of the association between FMD and FMV (Martelli, 2011). Mosaic-like symptoms were also observed in at least five fig trees of cv. Bicariña, Cotia, Morada and da Libra that were PCR-negative for FMV, but contained mixtures of three or four of the other viruses they were tested for. This finding further supports the complex nature of FMD, in whose aetiology FMV plays a significant but likely not an exclusive role (Martelli, 2011). Whereas no virus-free plants were found in the collection plot of ICIA at Tenerife, one tree each of cvs Bravera cotia, Blanca de invierno and Bicariña negra from private gardens did not show visible symptoms at the time of the survey and did not contain any of the five viruses. These plants, after a confirmatory round of additional assays, may represent potential sources of material for propagation in the framework of a sanitary improvement programme which should be established in the Canary Islands.

REFERENCES Bradfute O.R., Whitmoyear R.E., Nault R.L., 1970. Ultrastructure of plant leaf tissues infected with mite borne vi-

Received April 4, 2011 Accepted June 21, 2011

ral-like particles. Proceedings of the Electron Microscopy Society of America 258: 178-179. Condit I.J., Horne W.T., 1933. A mosaic of the fig in California. Phytopathology 23: 887-897. Elbeaino T., Digiaro M., De Stradis A., Martelli G.P., 2006. Partial characterization of a closterovirus associated with a chlorotic mottling of fig. Journal of Plant Pathology 88: 187-192. Elbeaino T., Digiaro M., De Stradis A., Martelli G.P., 2007. Identification of a second member of the family Closteroviridae in mosaic-diseased figs. Journal of Plant Pathology 89: 119-124. Elbeaino T., Digiaro M., Alabdullah A., De Stradis A., Minafra A., Mielke N., Castellano M.A., Martelli G.P., 2009. A multipartite single-stranded negative-sense RNA virus is the putative agent of fig mosaic disease. Journal of General Virology 90: 1281-1288. Elbeaino T., Heinoun K., Digiaro M., Martelli G.P., 2010. Fig mild mottle-associated virus, a novel closterovirus infecting fig. Journal of Plant Pathology 92: 165-172. Flock R.A., Wallace J.M., 1955. Transmission of fig mosaic by the eriophyid mite Aceria ficus. Phytopathology 45: 52-54. Foissac X., Svanella-Dumas L., Gentit P., Dulucq M.J., Candresse T., 2001. Polyvalent detection of fruit tree Tricho-, Capillo- and Foveavirus by nested RT-PCR using degenerated and inosine containing primers (DOP RT-PCR). Acta Horticulturae 550: 37-43. Gattoni G., Minafra A., Castellano M.A., De Stradis A., Boscia D., Elbeaino T., Digiaro M., Martelli G.P., 2009. Some properties of Fig latent virus 1, a new member of the family Betaflexiviridae. Journal of Plant Pathology 91: 543-552 Martelli G.P., 2011. Fig mosaic disease and associated viruses. In: Hadidi A., Barba M., Candresse T., Jelkmann W. (eds). Virus and Virus like Diseases of Pome and Stone fruits, pp. 281-287. APS Press, St. Paul, MN, USA. Slykhuis J.T., 1973. Viruses and Mites. In: Gibbs A.J. (ed). Viruses and Invertebrates, pp. 391-405. North Holland Publishing Co., Amsterdam, The Netherlands.

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