Symptoms and Diagnosis
In vanilla, potyviruses cause a more or less pronounced mosaic and deformation of leaf blades sometimes associated with necrotic lesions (Figure 7.2). The mosaic and necrosis can also be visible on the stem. These symptoms are particularly spectacular on the young leaves and are much attenuated on older leaves. In Reunion Island, potyvirus symptoms were most obvious during the cool months with a remission of symptoms observed during the hot season (Benezet et al., 2000; Leclercq Le Quillec et al., 2001). The impact of the viruses on flowering and fruiting of the vanilla vines has not been established, although some reduction in growth can be inferred from the severe mosaic and deformation, affecting the shoots. Field observations revealed that the virus particles were unevenly distributed in the plant and symptomatic vines may test negative in ELISA (Leclercq Le Quillec et al., 2001). As a consequence, visual observation of mosaic on leaves and ELISA tests are complementary to assess potyvirus in vanilla plots, none of the technique used alone being sufficiently reliable for diagnosis.
FIGURE 7.2 (See color insert following page 136.) Potyvirus symptoms on leaves of V. planifolia (a = BYMV-Réunion, b = BCMV-Madagascar, c = necrotic strain of WMV-Tonga) and V. tahitensis (d = WMV-FP, e = DsMV-FP) and on stem (f = WMV-FP).
Several molecular techniques are available to diagnose potyvirus infection of vanilla, at the generic or specific level. The generic anti-potyvirus monoclonal antibodies (Jordan and Hammond, 1991), commercialized by Agdia (USA), proved to be efficient in detecting all the potyvirus species identified in vanilla by Indirect Simple Sandwich ELISA. Likewise, several motifs conserved in the genome of potyviruses have been used to design a number of degenerate primers for RT-PCR amplification of the corresponding sequence in the potyviruses genome (Chen et al., 2001; Colinet et al., 1998; Gibbs and Mackenzie, 1997; Ha et al., 2008; Langeveld et al., 1991; Marie-Jeanne et al., 2000; Pappu et al., 1993). Both techniques can be used to assess the potyvirus status of material but do not specifically identify the potyvirus species detected.
For potyviruses, specific identification by serology is unreliable because of the numerous cross reactions between virus species (Shukla et al., 1994). To overcome this problem a simple one tube, one-step, RT-PCR assay using degenerate primers followed by direct sequencing of a short fragment can be used (Grisoni et al., 2006). As RT-PCR and sequencing are getting easier and cheaper technologies, this method can be used in epidemiological surveys requiring high throughput. Microarray technologies, which are a promising way to get broad spectrum, specific, and sensitive detection tool for potyviruses, are under development (Boonham et al., 2007; Wei et al., 2009).
Epidemiology
Potyviruses are responsible for major losses of many economically important crops because they spread readily in the fields. Several potyviruses are seed transmitted, most are mechanically transmitted (albeit inefficiently), but all are efficiently transmitted by aphids in a nonpersistent manner (Brunt, 1992).
VanMV was readily transmitted from V. tahitensis to V. pompona using the peach aphid Myzus persicae (Wisler et al., 1987). Experimental transmission tests of WMV-Tonga from infected to healthy Nicotiana clevelandii using Aphis gossypii was successful but transmission tests involving V. planifolia as a donor plant with the same aphid had failed (Pearson and Pone, 1988; Pearson et al., 1990; Wang and Pearson, 1992; Wang et al., 1993, 1997). However, several field data on potyvirus infections support the role of aphids in the spread of potyviruses in vanilla plots (Richard et al., 2009). Successful aphid transmission trials were consistent with a nonpersistent mode of transmission. In nonpersistent transmission, a few seconds or minutes are sufficient for virus acquisition or virus inoculation by the vector in feeding probes. It is therefore not surprising to observe heavy potyvirus infection in the absence of established aphid had colonies on vanilla (excepting Cerataphis orchidarum, for which only wingless forms have been described and which is not considered an efficient virus vector).
A few potyviruses are transmitted by seeds, notably CABMV in several leguminous species (Gillaspie, 2001), BCMV in some Phaseolus and Vigna cultivars, and BYMV in several leguminous species (Aftab and Freeman, 2006). In addition, BCMV is transmitted by bean pollen (Card et al., 2007). DsMV, WMMV, and WVMV are not known to be seed transmissible, but it has been suggested that seed transmission might be more common than currently recognized particularly within the BCMV subgroup (Gibbs et al., 2008a).
Seed transmission of viruses has epidemiological implications through intercrop-ping and weed management. In the field, primary inoculum of potyvirus may come from infected vanilla cuttings or from surrounding plants, either perennial plants or annual weeds, in which the virus can overwinter when it is transmitted by seeds. From this primary inoculum, the potyvirus is then disseminated in the plot by aphids. Molecular similarity between isolates infecting vanilla and weeds provides support to the passage of viruses from one species to another. For instances, in Madagascar, BCMV isolates were identified in vanilla and in the bordering weed Senna sp. (Leguminosae), which were 100% identical in CP gene sequences (Grisoni et al., 2006). In Reunion Island, a rapid outbreak of CABMV in a recently planted vanilla plot using virus-free cutting was correlated to the intercropping with Vigna unguicu-lata in which CABMV is seed transmitted. On the contrary, the use of Gliricidia sepium, a legume tree frequently used as support in vanilla plots and which is, because of frequent pruning, heavily infested by Aphis craccivora aphids (an effi-cient vector of BCMV subgroup of viruses) does not seem to increase potyvirus incidence in vanilla plots compared to when aphid-free support trees are used, such as Leucena glauca (Grisoni et al., 2004). However, in the presence of other virus sources the high aphid populations could become an issue.
Control
Epidemiological data showed that severe potyvirus outbreaks occur primarily in intensive vanilla farming system and may originate from a diversity of virus species involving a diversity of host species, virus reservoirs, and probably aphid species.