Owing to the high stability of the virion and its elevated concentration in host tissues, CymMV is readily transmitted by mechanical means (Jensen and Gold, 1955). It can therefore spread efficiently in the field or in green houses in the absence of specific vectors, solely as a result of virus from infected sap entering micro injuries in epidermal cells of the host. This has been specifically demonstrated for vanilla in shade house programs in Reunion Island (Leclercq Le Quillec et al., 2001) and FP (Grisoni et al., 2004). CymMV monitoring in these shade houses showed a rapid spread in large clusters primarily along the rows. These results supported the assumption that CymMV is introduced into shade houses via infected cuttings collected in the field, and then spread from plant to plant mainly by cultural practices (artificial pollination, looping) and possibly by root anastomosis between adjacent vines.
It is most important for vanilla growers to appreciate that the absence of symptoms increases the probability of both primary and secondary dissemination of this virus, and therefore the importance of planting material that has been certified free of the virus.
Control
In the absence of curative means or resistant commercial vanilla varieties, prophylaxis is the only approach to avoid CymMV losses in vanilla crops. Since humans are the principal vector of the virus (by planting infected cutting or as a mechanical transmission agent) a simple strategy combining plant phytosanitary certification and good management practices is sufficient to prevent the disease. This was successfully implemented in Reunion Island and FP in new plantation programs at the beginning of the 2000s (Benezet et al., 2000; Richard et al., 2009).
A basic requirement of phytosanitary certification is the need for virus-free material, which should be multiplied according to a specific protocol. A healthy starting material can generally be found within the cultivated stock but where a particular variety is required for which only infected germplasm is available, plants need to be cured of the virus. Vanilla is a clonal crop with a high degree of heterozygosity (see Chapter 2), and although seedlings give rise to virus-free vanilla (Jensen and Gold, 1955; Yuen et al., 1979), they are not true to type. Virus elimination has been achieved for several orchid species by chemotherapy in vitro (Albouy et al., 1988; Lim et al., 1993a) and similar strategies should be transferable to vanilla.
Genetic resistance or tolerance is particularly desirable when prophylaxis fails to provide sufficient control of viral disease. Apart from the resistance described in V. pompona, no natural resistance is available in V. planifolia. Pathogen-derived acquired resistance to CymMV has been bio-engendered in some orchid species (Chang et al., 2005; Liao et al., 2004; Lim et al., 1999), and although this strategy has not yet been developed for vanilla, it is theoretically feasible, since regeneration of vanilla protoplasts has been recently achieved (Minoo et al., 2008).
Potyviruses
The genus Potyvirus represents the second largest group of plant pathogenic viruses (about 25% of all known plant viruses species), making the potyviruses a worldwide agricultural concern (Fauquet et al., 2005). A potyvirus causing leaf distortion and mosaic in V. tahitensis vines was identified in 1986 in FP (Wisler et al., 1987). This virus, named Vanilla mosaic virus (VanMV), was shown to be serologically, then genetically, related to Dasheen mosaic virus (DsMV) (Farreyrol et al., 2006; Wang and Pearson, 1992; Wisler et al., 1987). Contemporaneously another potyvirus causing necrosis in V. planifolia was identified in 1986 in Tonga and tentatively named Vanilla necrosis virus (VNV) before being characterized as a strain of Watermelon mosaic virus (WMV-Tonga) (Pearson and Pone, 1988; Pearson et al., 1990). The biological properties of WMV-Tonga have been well characterized and its full CP gene sequence determined (Pearson et al., 1990; Wang et al., 1993). Wang and Pearson (1992) demonstrated that WMV-Tonga and DsMV-Vanilla were distinct viruses. DsMV-Vanilla was subsequently detected serologically in V. tahitensis in the Cook Islands, and in V. planifolia in Fiji and Vanuatu (Pearson et al., 1993), and WMV was detected in V. planifolia in FP (Grisoni et al., 2004). Several potyvirus isolates that did not react with either WMV or DsMV antisera were also reported (Grisoni et al., 2004; Pearson, 1997; Pearson et al., 1993), suggesting that other poty-viruses were present in vanilla crops.
The Viruses
The Potyvirus genus (family Potyviridae) encompasses 129 recognized species plus about 15 tentative species (Carstens and Ball, 2009; Fauquet et al., 2005). Potyviruses are found worldwide and infect more than 30 plant families, although individual viruses often have a restricted host range. They have flexuous, nonenveloped, rod-shaped particles (680–900 nm long, 12–15 nm in diameter) that contain a positive-sense single-stranded RNA molecule (Hull, 2002). The monopartite genome is about 10 kb in size and contains a single ORF that encodes a polyprotein of 3000–3300 amino acids. The polyprotein is cleaved co- and post-translationally by three virus-encoded proteinases, into 10 mature proteins, most of which are multifunctional (Adams et al., 2005a; Revers et al., 1999; Urcuqui-Inchima et al., 2001). An extensive study of gene diversity within the family Potyviridae (Adams et al., 2005b) concluded that the cytoplasmic inclusion (CI) protein gene was superior for potyvirus identification when using only a subportion of the genome. Although less informative than the CI, the CP gene can also efficiently separate the species, the demarcation criterion being defined as 76–77% nt identity by these authors.
The CP gene analysis from 36 vanilla samples collected in the Indian Ocean and Pacific regions between 1997 and 2005 added five potyvirus species to the two previously described infecting vanilla, namely: Bean common mosaic virus (BCMV), Cowpea aphid-borne mosaic virus (CABMV), Wisteria vein mosaic virus (WVMV), Bean yellow mosaic virus (BYMV), and Ornithogalum mosaic virus (OrMV) (Grisoni et al., 2006).
Remarkably, among the seven species that infect vanilla, five (BCMV, CABMV, DsMV, WMV, and WVMV) belong to the BCMV subgroup, which preferentially infect leguminous crops and weeds. They were detected in Reunion Island (BCMV and CABMV), in Madagascar (BCMV), in Mauritius (CABMV), in FP (BCMV, DsMV, and WMV), in Tonga (WMV), and in Samoa (WVMV). The two other species, BYMV and OrMV, were only detected in Reunion Island. The mosaic inducing potyvirus reported in India (Bhai et al., 2003; Bhat et al., 2004; Thomas et al., 2002) were not identified but may involve BYMV as suggested by the two GenBank accessions (AY845011 and AY845012) originating from vanilla material collected in Karnataka (India). Potyvirus-like symptoms have also been reported in Papua New Guinea (Kokoa, 2000) and in Puerto Rico (Childers and Cibes, 1948).
The DsMV strains infecting vanilla were at first tentatively referred to as VanMV because they showed only distant serological relationship to dasheen-infecting strains of DsMV and because of the failure of cross-inoculation between the strains originating from the two hosts. Subsequently, the nucleotide analysis of the 3′ end of the genome of two VanMV isolates (from Cook Islands [CI] and FP [FP]) confirmed that they should be classified as DsMV strains, since they shared more than 78% identities, notably in the core CP (Farreyrol et al., 2006). Interestingly, the DsMV-Vanilla [CI] and DsMV-Vanilla [FP] were as divergent from each other as from published DsMV-dasheen (Colocasia esculenta) strains (Farreyrol et al., 2006). However, recently obtained sequences for dasheen isolates from Cook Islands and FP were very similar to vanilla isolates from the same country (M. Pearson et al., unpubl. data). Since dasheen has been grown on these islands for much longer than vanilla, this suggests that “virus jump” from dasheen to vanilla may have occurred independently in the two Pacific Islands. In addition, the DsMV-Vanilla [FP] showed unusual features which makes it a particular virus among potyviruses (Farreyrol et al., 2006): a DVG aphid transmission motif (instead of the more common DAG motif) upstream of an unusual stretch of amino acid repeats (GTN) typical of natively unfolded proteins and an uncommon NIb/CP proteolytic cleavage site (Q//V).