# An analysis of viruses associated with cassava mosaic virus disease in three Angolan provinces

S. Matic, A.T. Pais da Cunha, Jeremy R. Thompson, Mark Tepfer

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S. Matic, A.T. Pais da Cunha, Jeremy R. Thompson, Mark Tepfer. An analysis of viruses associated with cassava mosaic virus disease in three Angolan provinces. Journal of Plant Pathology, 2012, 94 (2), pp.443-450. 10.4454/JPP.FA.2012.043. hal-01191289

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SHORT COMMUNICATION

AN ANALYSIS OF VIRUSES ASSOCIATED WITH CASSAVA MOSAIC DISEASE IN THREE ANGOLAN PROVINCES

S. Matic¹,²*, A.T. Pais da Cunha³,⁴, J.R. Thompson¹,⁵ and M. Tepfer¹,⁶,⁷

¹Plant Virology Group, ICGEB Biosafety Outstation, Via Piovega 23, 31056 Ca'Tron di Roncade, Italy

²Istituto di Virologia Vegetale, CNR, Strada delle Cacce 73, 10135 Torino, Italy

³Dipartimento Territorio e Sistemi Agro-Forestali - Patologia Vegetale, Università degli Studi di Padova, Agripolis, Viale dell'Università, 16, 35020 Legnaro (PD), Italy

⁴Departamento de Agronomia - Instituto Superior Politécnico do Kwanza sul, Rua 12 Novembro, Sumbe, Angola

⁵Department of Plant Pathology and Plant-Microbe Biology, 334 Plant Science Building, Cornell University, Ithaca, NY 14853, USA

⁶INRA, UMR1318, Institut Jean-Pierre Bourgin, INRA-Versailles 78026 Versailles cedex, France

⁷INRA, UR407, Station de Pathologie Végétale, INRA-Avignon, 84140 Montfavet cedex, France

*Present address: Centre for Agro-Environmental Innovation (AGROINNOVA), Università degli Studi di Torino, Via Leonardo da Vinci 44, 10095 Grugliasco (TO), Italy

SUMMARY

Seventeen cassava plants showing strong cassava mosaic disease symptoms were sampled from three Angolan provinces. The DNA-A component of two isolates was amplified by rolling circle amplification (RCA) and characterized. The complete DNA-A of 2,778 nucleotides from sample AOS showed very high similarity to *African cassava mosaic virus* (ACMV), and that of 2,799 nucleotides from sample AO7 showed high identity to *East African cassava mosaic virus* (EACMV), particularly to EACMV-Uganda2 Severe strain. This represents the first report of complete ACMV and EACMV DNA-A sequences from Angola. PCR followed by direct sequencing of PCR products of a portion of DNA-A and DNA-B detected ACMV or EACMV in all 17 cassava plants. None of the five other cassava mosaic virus species were found. The presence of a mixed infection of ACMV and EACMV-Uganda2 Severe strain, associated with cassava mosaic disease pandemic, highlights the need for stricter controls on the importation of infected cassava stem cuttings from neighbouring countries and further investigations on the sanitary status of cassava plants in the presently unsurveyed provinces of Angola.

*Key words:* Begomovirus, RCA, DNA sequencing, phylogenetic analyses.

Cassava mosaic disease (CMD) is the most important
viral disease affecting cassava (Manihot esculenta
Crantz) in Africa (Thresh *et al.*, 1994; Legg *et al.*, 2006).
CMD is caused by several whitefly-transmitted viruses
belonging to the genus Begomovirus (family Geminiviridae) which have circular, single-stranded DNA genomes

(Briddon and Markham, 1995). The majority of begomoviruses have bipartite genomes, termed DNA-A and DNA-B (Stanley et al., 2004). The most recent taxonomic approach recognises seven African cassava mosaic geminivirus species: *African cassava mosaic virus* (ACMV), *East African cassava mosaic virus* (EACMV), *East African cassava mosaic Cameroon virus* (EACMCV), *East African cassava mosaic Kenya virus* (EACMKV), *East African cassava mosaic Malawi virus* (EACMMV), *East African cassava mosaic Zanzibar virus* (EACMZV), and *South African cassava mosaic virus* (SACMV) (Fauquet et al., 2008).

After the first report of the severe form of CMD in
Uganda, a novel recombinant begomovirus named
EACMV-Uganda2 Severe, which we will abbreviate as
EACMV-UG, has been associated with the severe CMD
pandemic (Deng et al., 1997; Zhou et al., 1997).
EACMV-UG was found either singly or in mixed infec-
tion with ACMV (Harrison et al., 1997; Pita et al., 2001;
Ntawuruhunga et al., 2007). The EACMV-UG genome
is distinguished from that of other EACMV strains by
the presence of a region in the coat protein (CP) AV1
gene of ca. 500 bp with high sequence identity to the
corresponding part of the ACMV genome.

Angola is one of the biggest cassava producers in
southern Africa. Food security of the country is influ-
enced by the success of cassava production, which is di-
rectly related with the presence of CMD and crop pro-
tection programmes. To our knowledge, there are only
two reports on viruses associated with CMD in Angola.
The presence of EACMV from one cassava plant in
southern Angola (Okavango region) was reported by
Berry and Rey (2001). More recently, using ELISA and
species-specific PCR, ACMV was determined to be the
predominant species, followed by EACMV and
EACMV-UG from the Cuanza Norte, Malanje and Uige
provinces of Angola (Lava Kumar *et al*., 2008). Despite
these studies, there is still a lack of data regarding the
molecular characterization and genomic variability of these viruses in the country, which would greatly assist in the development of control strategies. The objective of this study was to analyze the presence of viruses in cassava plants exhibiting CMD symptoms, and to estimate the genetic diversity among the virus isolates associated with the disease in three Angolan provinces.

Seventeen cassava plants showing strong CMD symptoms were sampled from farms in Cabo Ledo (Bengo province), Canjala (Benguela province), and Porto-Amboim, Sumbe and Uku Seles (Cuanza Sul province) during January 2009 (Table 1, Fig. 1). CMD symptoms were observed in all surveyed orchards. Plants were stunted, and leaves were mottled, distorted, and smaller. Samples were taken from different native and imported cultivars, such as Precose de Angola, Mundele Paço, Ngana rico, Thiti, TMS 30142, and TMS 40125.

Total nucleic acid (TNA) was extracted from 100 mg of tissue (leaf or phloem scrapings) according to Foissac *et al.* (2001). Circular DNA was amplified using the Ilustra TempliPhi 100 Amplification Kit (GE Healthcare, USA) following the manufacturer's instructions. RCA products were digested by the restriction endonucleases

*Eco*RI and *Bam*HI (New England Biolabs, USA). A fragment of approximately 2.8 kb was amplified from all Angolan samples, the size expected for the DNA-A of begomoviruses. The 2.8 kb-band from sample AOS (obtained with *Eco*RI) and AO7 (obtained with *Bam*HI) was excised and purified using the QIAquick Gel Extraction Kit (Qiagen, USA). Purified products were cloned into the *Eco*RI or *Bam*HI site of a previously digested pBluescript KS (+) vector (Stratagene, USA), then transformed into *Escherichia coli* DH5α cells by electroporation. Plasmids containing the desired inserts were extracted by Jetprep Plasmid Miniprep Kit (Genomed GmbH, Germany). DNA sequences of the selected recombinant plasmids were obtained by automatic sequencing at BMR (Padova, Italy). Multiple sequence alignment of nucleotide (nt) and amino acid (aa) sequences and identification of open reading frames (ORFs) were done using the program AlignX (Vector NTI Suite V 5.5, InforMax, USA) with the Clustal W algorithm (Thompson *et al.*, 1994). Detection of possible recombinants was carried out using the RDP3 program (Martin *et al.*, 2010). Phylogenetic analysis was performed using PAUP 4.0 (Swofford, 2001), with the

<figure><img src="image_3.png" /><figcaption>**Fig. 1.** A map of Angola showing the localities in Bengo, Cuanza Sul and Benguela provinces where cassava plants were sampled.</figcaption></figure>
<table><caption>**Table 1.** Angolan cassava isolates used for molecular characterization by RCA and PCR.</caption><thead><tr><th rowspan="2">Isolate code</th><th rowspan="2">Province</th><th colspan="2">ACMV</th><th colspan="2">EACMV</th></tr><tr><th>Accession No. DNA-A (AC2/AC1)</th><th>Accession No. DNA-B (BV1/BC1)</th><th>Accession No. DNA-A (AC2/AC1)</th><th>Accession No. DNA-B (BC1)</th></tr></thead><tbody><tr><td>AO1</td><td>Bengo</td><td>GU580899</td><td></td><td></td><td></td></tr><tr><td>AO2</td><td>Bengo</td><td>GU580900</td><td></td><td></td><td>GU580925</td></tr><tr><td>AO3</td><td>Bengo</td><td>GU580901</td><td>GU580915</td><td>GU580911</td><td>GU580926</td></tr><tr><td>AO4</td><td>Bengo</td><td></td><td></td><td></td><td></td></tr><tr><td>AO5</td><td>Bengo</td><td>GU580902</td><td>GU580916</td><td></td><td>GU580927</td></tr><tr><td>AO7</td><td>Bengo</td><td>GU580903</td><td></td><td>JN941177*</td><td></td></tr><tr><td>AO9</td><td>Bengo</td><td>GU580904</td><td></td><td></td><td></td></tr><tr><td>AO10</td><td>Bengo</td><td>GU580905</td><td></td><td></td><td></td></tr><tr><td>AOUN</td><td>Benguela</td><td>GU580910</td><td></td><td></td><td>GU580934</td></tr><tr><td>AOA</td><td>Cuanza Sul</td><td>GU580906</td><td>GU580917</td><td></td><td>GU580928</td></tr><tr><td>AOE</td><td>Cuanza Sul</td><td></td><td>GU580918</td><td>GU580912</td><td>GU580929</td></tr><tr><td>AOH</td><td>Cuanza Sul</td><td>GU580907</td><td>GU580919</td><td></td><td>GU580930</td></tr><tr><td>AOJ</td><td>Cuanza Sul</td><td>GU580908</td><td>GU580920</td><td></td><td></td></tr><tr><td>AOK</td><td>Cuanza Sul</td><td></td><td>GU580921</td><td>GU580913</td><td>GU580931</td></tr><tr><td>AON</td><td>Cuanza Sul</td><td>GU580909</td><td>GU580922</td><td></td><td>GU580932</td></tr><tr><td>AOO</td><td>Cuanza Sul</td><td></td><td>GU580923</td><td>GU580914</td><td>GU580933</td></tr><tr><td>AOS</td><td>Cuanza Sul</td><td>GU580897*</td><td>GU580924</td><td></td><td></td></tr></tbody></table>

best model being selected by qModeltest (Posada, 2009).

A set of primers (ACMV21for and ACMV21rev) was designed for the detection of all known African cassava begomoviruses (Table 2). They were based on the sequences of all isolates available in GenBank corresponding to nts 1,447-1,998 of DNA-A of ACMV isolate GU580897 as determined by BLASTx. Since these primers allowed the amplification of DNA-A sequences of all known African cassava begomoviruses, specific primers were designed for the discrimination between ACMV and EACMV in DNA-B: (i) ACMVB1 and ACMVB2 for ACMV, corresponding to nts 746-1,373 of isolate AF259895; and (ii) ACMVB1 and EACMB2 for EACMV, corresponding to nts 1,343-1,969 of isolate AF230375 (Table 2).

PCR was done with AccuPrime™ Taq DNA Polymerase (Invitrogen, USA) according to the manufacturer's instructions. PCR conditions were 94°C for 5 min for denaturation, followed by 35 cycles at 94°C for 1 min, 55°C for 1 min and 72°C for 1 min, and a final extension step of 5 min at 72°C. PCR products were analysed on a 1.2% agarose/1X TAE gel stained with ethidium bromide, purified and extracted using the Jetquick Gel Extraction Spin Kit (Genomed GmbH, Germany) and sequenced directly at BMR (Padova, Italy). Sequenced PCR products were deposited in GenBank with accession numbers shown in Table 1.

One RCA clone each was obtained from samples AOS and AO7. BLAST analysis of the sequence of the

AOS clone (accession No. GU580897) revealed a very high identity to ACMV DNA-A, showing the highest nt identity (96%) with an ACMV isolate from Uganda (ACMV-[UG:Mpi:56:02], accession No. AM502339). The full-length genomic DNA-A of ACMV-AOS was 2,778 nt long. It comprised six ORFs, two (AV2 and AV1) in the virion sense and four (AC3, AC2, AC1 and AC4) in the complementary sense that were separated by an intergenic region (IR) containing the conserved nona-nucleotide motif (TAATATTAC) essential for the initiation of rolling circle replication (Chatterji *et al.*, 1999; Rojas *et al.*, 2005).

The sequenced full-length DNA-A of the AO7 clone (2,799 nt, accession No. JN941177) showed high identity to EACMV DNA-A. This sequence contained AV2 and AV1 genes in the virion sense, and AC5, AC3, AC2, AC1 and AC4 genes in the complementary sense. It showed the highest nt identity (99%) with an EACMV isolate from cassava in Burkina Faso (accession No. FM877474) and an EACMV isolate from *Pueraria ja-vanica* from the Democratic Republic (DR) of Congo (accession No. FN435279), which both belong to the severe EACMV-UG strain. Furthermore, EACMV-AO7, when compared with the reference Z83257 isolate of EACMV-UG, showed 99% nt identity in the recombinant CP gene, which confirmed its affiliation to EACMV-UG.

In analysing possible recombination events, there was evidence of multiple breakpoints in EACMV-AO7, with all but one of the putative parents derived from the

<footnote>*Sequences in bold were obtained by RCA and subsequent cloning, all other sequences were obtained by PCR.</footnote>
principal clades of EACMV. According to the RDP3 program, a region from 544 to 1,008 nts of the genome could plausibly have been derived from the ACMV isolate AF126800 (Fig. 2a). Interspecies genetic exchange is also in evidence for ACMV-AOS, in which a short stretch from 2,123 to 2,298 nts was found to be closest to EACMKV isolate AJ717582 (Fig. 2b). These proposed recombinant structures were a signature for all isolates within the same monophyletic group as shown in Fig. 3.

A primer pair (ACMV21for/rev) was designed from the sequences of a portion of DNA-A (AC3-AC2-AC1) of all African cassava mosaic begomoviruses (Table 2), which allowed the detection of ACMV or EACMV in all 17 Angolan samples after sequencing of the 467 bp PCR products, including a portion of AC2-AC1 (Table 1). Sequencing PCR products confirmed the presence of ACMV in sample AOS, whereas it detected ACMV in sample AO7, in which EACMV had already been found by sequencing the RCA product. In the 15 re-

a JN941177 AO7_AGO_EACMV

<figure><img src="image_4.png" /></figure>

<table><thead><tr><th></th><th>AF259896_CIV</th><th>AF259896_CIV</th><th>AF126800_UGA</th></tr></thead><tbody><tr><th>RDP</th><td>3.3 x 10<sup>-42</sup></td><td>9.1 x 10<sup>-20</sup></td><td>2.2 x 10<sup>-68</sup></td></tr><tr><th>GENECONV</th><td>7.3 x 10<sup>-36</sup></td><td>3.9 x 10<sup>-17</sup></td><td>6.9 x 10<sup>-66</sup></td></tr><tr><th>MaxChi</th><td>5.4 x 10<sup>-28</sup></td><td>3.4 x 10<sup>-23</sup></td><td>3.8 x 10<sup>-19</sup></td></tr><tr><th>Chimaera</th><td>6.9 x 10<sup>-30</sup></td><td>5.5 x 10<sup>-28</sup></td><td>2.5 x 10<sup>-19</sup></td></tr><tr><th>3Seq</th><td>1.0 x 10<sup>-80</sup></td><td>1.7 x 10<sup>-72</sup></td><td>6.4 x 10<sup>-68</sup></td></tr></tbody></table>

b GU580897 AOS_AGO_ACMV

<figure><img src="image_5.png" /></figure>

<table><tr><th>RDP</th><td>4.1 x 10<sup>-9</sup></td></tr><tr><th>GENECONV</th><td>4.9 x 10<sup>-6</sup></td></tr><tr><th>MaxChi</th><td>9.0 x 10<sup>-9</sup></td></tr><tr><th>Chimaera</th><td>1.4 x 10<sup>-6</sup></td></tr><tr><th>3Seq</th><td>2.6 x 10<sup>-6</sup></td></tr></table>

**Fig. 2.** Evidence of recombination in isolates (a) EACMV-AO7 (accession no. JN941177) and (b) ACMV-AOS (accession No. GU580897). A simplified linearized map of each isolate is depicted with the position of the crossover nucleotides for each putative recombination region (light grey boxes) indicated. The floating boxes below correspond to the minor parent donor, the name of which is placed alongside or underneath for reference. The putative major parent donor names are placed above their associated fragments. The probability values for the five main methods employed by the RDP3 program are shown in the boxes with the relevant putative minor donor fragment (when relevant) above each column. Note that for ACMV-AOS no major parent was identified. Given the circular structure of the genomes the donation of AF259896_CIV_EACMCV to EACMV-AO7, though depicted as fragmented is continuous through nts 2799 and 1.
<figure><img src="image_6.png" /><figcaption>Fig. 3</figcaption></figure>

**Fig. 3.** Neighbour-joining phylogenetic tree of the full-length DNA-A sequences of Angolan ACMV and EACMV virus isolates (highlighted) at the nucleotide level, compared to isolates of the other African cassava mosaic virus species. Numbers at the branches refer to the bootstrap values for 100 replicates, which if below 70 were collapsed. Distance (indicated by the bar) refers to the number of nucleotide changes per base. Three letter codes at the end of each accession reference refers to the country of origin: Angola-AGO, Burkina Faso-BFA, Cameroon-CMR, Democratic Republic of Congo-COD, Ivory Coast-CIV, Kenya-KEN, Madagascar-MDG, Malawi-MWI, Nigeria-NGA, South Africa-ZAF, Tanzania-TZA, Uganda-UGA, Zimbabwe-ZWE. Groupings for each virus species is shown by the brackets on the left, with all 'East African' species (EACMV, EACMCV, EACMKV, EACMV, EACMZ) grouped as EACMV. Relevant putative recombinant donor molecules (major parents large and bold) for EACMV-AO7 and ACMV-AOS, as determined by RDP3 (see Fig. 2) are marked with a star and hatch respectively. Cross hatches on the branches mark monophyletic groups with similar proposed recombination structures, as depicted in Fig. 2.
<table><caption>**Table 2.** Primers used for characterization of Angolan cassava isolates affected by Cassava mosaic disease.</caption><thead><tr><th>Primer (5'-3')</th><th>Gene target region</th><th>Expected size of PCR amplicon</th><th>Target viruses</th></tr></thead><tbody><tr><td>ACMV21for:<br>(GCAGTGATGAGTTCCCCGGTGCG) sense</td><td rowspan="2">AC3/AC2/AC1</td><td rowspan="2">552 bp</td><td rowspan="2">Seven African cassava mosaic viruses<sup>a</sup></td></tr><tr><td>ACMV21rev:<br>(ATTCCGCTGCGCGGCCATGGAGACC) antisense</td></tr><tr><td>ACMVB1:<br>(TCGGGAGTGATACATGCGAAGGC) sense</td><td rowspan="2">BV1/BC1</td><td rowspan="2">628 bp</td><td rowspan="2">ACMV</td></tr><tr><td>ACMVB2:<br>(GGCTACACCAGCTACCTGAAGCT) antisense</td></tr><tr><td>EACMVB1:<br>(GAGGTCCCTTTCTGGATACTGAGTCGG) sense</td><td rowspan="2">BC1</td><td rowspan="2">627 bp</td><td rowspan="2">EACMV</td></tr><tr><td>EACMVB2:<br>(GACAATCGCGTCAGCCTTGAAGACG) antisense</td></tr></tbody></table>

maining Angolan samples, ACMV was detected in 11, and EACMV in four samples. None of the five other cassava mosaic virus species were found.

DNA-B primer pairs were designed for specific detection of ACMV (ACMVB1/B2) or EACMV (EACMVB1/B2) (Table 2). This allowed detection of DNA-B of either virus in 12 Angolan samples, which was confirmed with direct sequencing of the PCR products (Table 1). Surprisingly, the DNA-B-specific primers failed to detect virus in five samples; probably consequent to genetic variability within the targeted DNA-B segment.

Both sets of primer pairs revealed the presence of ACMV and EACMV in single (six isolates) and mixed infections (eleven isolates) (Table 1). ACMV was predominant in single infections. In some mixed infections, ACMV and EACMV presence was detected only for the DNA-A or DNA-B component, which might be evidence of trans-replication between ACMV DNA-A and EACMV DNA-B, as already reported in natural infections (Pita *et al.*, 2000).

Phylogenetic analysis of nt (Fig. 3) or aa sequences (not shown) of the full-length DNA-A of ACMV-AOS and EACMV-AO7, together with all representative African cassava mosaic begomovirus sequences available in GenBank, grouped ACMV-AOS with other ACMV isolates, while EACMV-AO7 grouped within EACMV, and was closest to an EACMV-UG isolate from Burkina-Faso.

Previous reports have demonstrated the presence of ACMV and EACMV in Angola (Berry and Rey, 2001; Lava Kumar *et al.*, 2008), which we have further confirmed in this work. We have sequenced EACMV isolates from north-western (Bengo) and central-western

(Cuanza Sul and Benguela) provinces of Angola, which showed the greatest identity with an EACMV-UG strain. The pandemic of severe CMD has spread from east and central Africa to west African countries (Ndunguru *et al.*, 2005; Legg *et al.*, 2006; Patil and Fauquet, 2009). DR Congo, which neighbours Angola on the north, has already been affected by the CMD epidemic, and EACMV-UG is present there (Monde *et al.*, 2010). Furthermore, the diffusion of EACMV-UG towards West Africa was evidenced from Burkina Faso by Tien-drébéogo *et al.* (2009). Since EACMV-AO7 from Angola shows the highest identities with EACMV-UG from DR Congo and Burkina Faso, it might further confirm the route of CMD pandemic movement from East to West Africa, either by long-distance distribution through infected stem cuttings, or by whitefly vector transmission. As EACMV-AO7 belongs to EACMV-UG and shares 98-100% nt identity with other characterized Angolan EACMV isolates, it suggests that all the Angolan EACMV isolates could be variants of this strain, but this can only be determined with certainty by sequencing the CP of these isolates. Our results confirm the CMD pandemic development estimation done by Legg *et al.* (2006), where northern Angola was predicted to be affected by CMD.

The EACMV isolates characterized in this work could represent recently established virus populations originating from the recombinant EACMV-UG, while the EACMV isolate described by Berry and Rey (2001) may represent a distinct introduction event. In contrast, the Angolan ACMV isolates identified here had highest identities with isolates originating from different countries (Uganda, Burkina Faso, DR Congo, Nigeria, and Ivory Coast) which could have resulted from different

<footnote><sup>a</sup>African cassava mosaic virus (ACMV), East African cassava mosaic virus (EACMV), East African cassava mosaic Cameroon virus (EACMCV), East African cassava mosaic Kenya virus (EACMKV), East African cassava mosaic Malawi virus (EACMMV), East African cassava mosaic Zanzibar virus (EACMZV), and South African cassava mosaic virus (SACMV).</footnote>
introduction events or from the fact that ACMV was introduced in Angola a long time ago during which evolutionary divergence occurred, causing its higher genetic diversity.

The high co-infection rate of ACMV and EACMV-UG found in this study (65%) fits well with the findings reported from neighbouring DR Congo (Monde et al., 2010) and the Republic of Congo (Ntawuruhunga et al., 2007). It was reported that after establishment of epidemic CMD, ACMV was replaced by the more virulent EACMV-UG, and that the proportion of mixed infections was significantly reduced (Legg and Thresh, 2000; Legg et al., 2006). The high presence of mixed infections and the prevalence of ACMV in single infections we found in Angola could indicate a still recent introduction of EACMV-UG to Angola that has not yet displaced ACMV.

These findings highlight the urgent need for large-scale field surveys of cassava affected by CMD in the presently unsurveyed provinces of Angola, along with epidemiological studies to clarify the significance of mixed infections between ACMV and EACMV-UG and the role of whitefly in the spread of CMD. The introduction of resistant cultivars in combination with rigorous phytosanitary measures have already been used to combat the disease in other pandemic-affected countries of Africa (Legg et al., 1999). The control of importation of infected cassava material is of crucial importance, since the movement of infected stakes increases the risk of the introduction of new cassava mosaic viruses, and new mixed infections, thus increasing the likelihood of recombination and reassortment between existing viruses.

In conclusion, cassava begomovirus diversity detected so far in Angola is consistent with the diversity known in neighbouring countries and suggests several introduction events of ACMV and a recent introduction of EACMV-UG.

## ACKNOWLEDGEMENTS

The authors wish to thank Dr. E. Noris and Dr. G.P. Accotto (Istituto di Virologia Vegetale del CNR, Torino, Italy) for final discussion and support in this work.

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