Distribution and multiplication of iris severe mosaic potyvirus in bulbous Iris in relation to metabolic activity : implications for ISMV detection

During cultivation of iris, several viruses may cause severe damage like yield reduction and discoloration of the plant. In commercial stocks in the Netherlands virtually all plants are infected with iris mild mosaic virus (IMMV) while iris severe mosaic virus (ISMV) and narcissus latent virus (NLV) can also be present at high incidence. ISMV and IMMV both belong to the genus Potyvirus of the largest plant virus family, the Potyviridae.As the quality of virus-free iris is superior and methods are available now to produce and grow virus-free iris for commercial practice, it is of great importance to control the spread of iris viruses. Therefore rapid and reliable assays for the detection of these viruses are needed. In the Netherlands such tests are being developed mainly at the Bulb Research Centre in Lisse. At the start of the research described in this thesis, detection of ISMV in the iris bulb was problematic, in contrast to that of IMMV It has been difficult to detect ISMV reliably in iris bulbs from lifting in late August to planting in October by means of ELISA or electron microscopy.The aim of the research was to develop a reliable detection protocol suitable for monitoring ISMV infection, and, to understand the behaviour of this virus in the iris plant, especially the bulb, with respect to the multiplication and movement of the virus in relation to the metabolic activity of the plant. Preliminary results concerning the development of a test protocol are presented in Chapter 2, and further elaborated in Chapter 3, 7 and 8. In Chapters 4, 6 and 7 the behaviour of ISMV in relation to the metabolic activity of the plant is elaborated. In Chapters 5 and 8 a further characterisation of ISMV is presented.In freshly-lifted bulbs secondarily infected with ISMV, the virus was not always detected in the basal plate and rarely in bulb scale tissue (Chapter 4), but it gradually became better detectable in the bulb scale tissue when bulbs were incubated during several months at a temperature of circa 17°C (Chapter 2). When a wounding method was applied on the iris bulb by cutting a slice of bulb scale tissue from a side of the bulb, ISMV became readily detectable in all bulbs, though only in tissue adjacent to the cut surface, if the cut bulbs were incubated for three weeks at an optimal temperature of 17-20°C (tested within a range of 5 to 30°C; Chapters 2 and 3). It was concluded that stress followed by a recovery period is favourable for an enhanced detection of the virus. Indeed, high temperature treatment, applied as an alternative stress, also gave rise to improved detection of ISMV (Chapter 3).To investigate whether the virus became better detectable by multiplication rather than by modification of the antigenicity of the coat protein, the levels of the viral antigen as well as those of the viral RNA were followed after wounding. From this analysis it was concluded that the increase of the virus titre was due to multiplication (Chapter 4). For the detection of the viral RNA, a cDNA done corresponding to a part of the 3'-terminal region of the ISMV genome was used. The availability of this clone led to the determination of the nucleotide sequence of the ISMV coat protein (CP) gene, thus allowing a definitive classification of the virus. Phylogenetic comparisons of potyviral CP sequences revealed that ISMV is a taxonomically distinct potyvirus not closer related to other bulbous or monocotyledonous infecting potyviruses than to other potyviruses. The sequence data also allowed to conclude that the CP is probably cleaved off from the NIb protein at an unusual glutamine acid-glycine (E/G) dipeptide cleavage site. Furthermore the N-terminus of the CP appeared to be only 15 amino acids long, being the shortest found among potyvirus CPs studied so far.Further research on the localisation of the virus after high-temperature treatment showed that the virus was well detectable in the bulb base and usually also in the vascular bundles and surrounding tissue. This suggested that the virus did spread from the basal plate towards the bulb scales. However when the wounded (cut) apical parts of infected, but in ELISA negative reacting, bulbs were incubated at an optimal recovery temperature, the virus became detectable in these upper parts of the bulbs (Chapter 4). Thus, virus must have been originally present in the scales, albeit at a very low and at non-detectable concentration. This provides another indication that multiplication is likely to be the main factor involved in the improved sensitivity of viral detection. It is, therefore, hypothesised that the multiplication is enhanced by increased metabolic activity after stress. A possible correlation between metabolism and ISMV multiplication was further investigated in Chapter 6, with oxygen uptake as a measure for the metabolic activity after application of wounding, high-temperature stress or ethylene treatment.An increased level of total oxygen uptake was found after wounding as well as high- temperature treatment, thus positively correlating with the enhancement of ISMV detection. Application of ethylene, an important plant hormone in relation to stress, caused a limited increase in respiration and a slight improvement of ISMV detectability. After wounding, the mitochondrial respiration, the residual respiration and the capacity of the alternative pathway had increased, while after high-temperature treatment there was mainly an increase in residual respiration measured. These findings suggest that increased production of metabolic intermediates, possibly by the pentose phosphate pathway, rather then an increase in energy is important for the observed stress-induced multiplication of ISMV in iris bulbs.For the development of a satisfactory test method, it is imperative that virus is reliably detected not only in secondarily infected bulbs but also in primarilyinfected bulbs. To obtain primarily infected bulbs, virus-free plants were mechanically inoculated with ISMV at different times during the growing season. At lifting the level of ISMV in primarily infected bulbs appeared to be dependent on the date of inoculation. Surprisingly, it was found that early infections were scarcely detectable in the bulbs in contrast to late infections. The later in the season infection took place, the better ISMV was detectable in (untreated) bulbs (Chapter 7). Wounding of these primarily infected bulbs generally resulted in an increased detection in bulbs of the early infected plants, but the virus titre in bulbs of late infections decreased. However, these infections were still reliably detectable. Another potential problem for implementation of the developed test for routine use could be the existence of differently reacting isolates of ISMV. In spite of causing slightly different symptoms and serological reactions, all could be detected by the wounding method (Chapter 8).The reason why ISMV is so difficult to detect in secondarily infected and in early primarily infected bulbs was investigated further in Chapter 7. The virus titre was monitored in the whole plant, including the bulb, during the growing season for both secondarily and primarily infected plants. The distribution of ISMV in the above-ground parts of secondarily infected as well as primarily infected plants correlated with the nutrient flow via the vascular system. This implied that above-ground parts of secondarily infected plants were totally infected while in primary infections the presence of virus was dependent on the time of infection: during an early infection virus still spread to the upper leaves of the plant and only later to the new bulb, while in late infected plants the virus was found mainly in the new bulb. However, in secondarily infected plants hardly any virus could be detected in the new bulbs at any time during the whole growing season. Besides, the detectability of ISMV in bulbs of early infected plants decreased considerably towards the end of the growing season. This might be explained by assuming that the plant develops a barrier at some time after infection blocking virus avenue to the bulb, or that the virus in secondarily and early primarily infected plants is no longer available for transport anymore (Chapters 7 and 9). It must be concluded that detection of ISMV in these secondarily and early primarily infected bulbs immediately after lifting is unreliable due to impeded import of virus into the bulb.