Study of the pathosystem: Begomovirus Bemisia tabaci I tomato on the South West islands of the Indian Ocean

Tomato yellow leaf curl virus (TYLCV) belongs to the genus Begomovirus within the Geminiviridae family, and is exclusively transmitted by the whitefly species Bemisia labaci (Hemiptera: Aleyrodidae) (Gennadius). It is an emerging virus which since the 1980's has globally spread over many tropical, subtropical, and Mediterranean countries. By now it has even reached such isolated places as La Réunion, an island situated in the south-western part of the Indian Ocean. Since its first introduction in 1997, the virus has spread quickly throughout this island, becoming a great constraint for tomato cultivation. At the same time an upsurge of whiteflies was reported on vegetable crops.For future control and prevention of begomovirus infestations on La Réunion further information on the identity and genetic properties of the introduced begomoviruses and on the whiteflies involved is urgenily needed. The aim of the studies presented in this thesis was, therefore, to investigate how new begomoviruses can potentially be introduced in La Reunion, which begomoviruses are currently present and which whitefly biotypes are involved in their spread. Also a start was made to search for useful resistance sources in a range of available wild Lycopersicon accessions.In Chapter 2, a potential way of unintentional introduction of new begomoviruses into an area free of begomoviruses was investigated- In La Réunion, and other areas, imported seedlings, ornamentals and all plant material are carefully checked at the custom for the presence of TYLCV or B. tabaci, due to their quarantine status, but not imported tomato fruits, as these were regarded not to represent a great risk. In view of the possibility that a potential import route had escaped attention, a survey of tomatoes imported into La Réunion had been made and it was demonstrated (Chapter 2) that more than 50% of the fruits contained TYLCV as determined by DNA blot analysis. Furthermore it was demonstrated that TYLCV can be acquired from this infected fruit by whiteflies and subsequently transmitted to healthy tomato plants. This successful transmission is consistent with the ELISA detection of TYLCV in different parts of vine tomato fruits and the 24 hours survival rate of80% of a whitefly population maintained on such fruits.Prior to 1997 hardly any virus problem was encountered in the tomato production in La Réunion. Since that year, the begomovirus TYLCV-MId was detected, causing yield losses of up to 80% in open field cultivation of susceptible cultivars. Since that year, surveys have been performed to monitor the disease incidence over the island. In Chapter 3 samplings from 1997 up to 2004 were studied in order to evaluate the mutation rate of this newIy introduced virus. The mutation frequency (number of mutations relative to the consensus, divided by the number of nucleotides sequenced), based on 2 regions of the genome, was estimated at 10 x 10" . This result confirmed that DNA plant viruses may have a mutation frequency equivalent to those obtained for plant RNA viruses. The low genetic diversity found in the initial population was consistent with a founder effect associated with a population bottleneck during the introduction of a new virus in an insular environment. It was hypothesized that the epidemic observed in 1997 in La Réunion was the result of only a single TYLCV introduction.The introduction of TYLCV on La Réunion illustrates that plant viruses are very successful in colonising new ecological niches. Realising the intensive trade of vegetables and fruits between all the South western islands of the Indian Ocean (SWIO), and that in 2001, a tomato virus symptom survey on the islands of Madagascar and Mayotte identified an association between B. tabaci and tomato plants displaying symptoms characteristic of begomoviruses, the survey for tomato begomoviruses was extended to these islands (Chapter 4). A first analysis of 1eaf samples collected during this survey indicated the presence of two potentially new Begomovirus species, which were called Tomato leafcurl Madagascar virus (ToLCMGV) and Tomato leafcurl Mayotte virus (ToLCYTV). Both biological and molecular properties of To LCMGV isolates from Morondova and Toliary (ToLCMGV-[ToI], -(Mor et al.)), ToLCYTV isolates from Dembeni and Kahani (ToLCYTV-[Dem], -[Kah]) and of a TYLCV isolate from La Réunion (TYLCV-MId[RE]) were determined (Chapter 4). Full-length DNA components of the five isolates from Madagascar, Mayotte, and La Réunion were cloned and sequenced and, with the exception of ToLCMG-[ToI], were shown to be both infectious in tomato resulting in virus transmissible by B, tabaci. Sequence analysis revealed that these viruses had genome organisations typical of monopartite begomoviruses and that both ToLCMGV and ToLCYTV belong to the African begomovirus but represent a distinct monophyletic group tentatively named the SWIO begomovirus group (Chapter 4). All of the SWIO isolates examined are apparently complex recombinants. None of the sequences within the recombinant regions close]y resembled that of any known non-SWIO begomovirus, suggesting an isolation of these virus populations.The upsurge of TYLCV observed in La Réunion in 1997 coincided with an increase of its insect vector on vegetable crops. As no such increase of B. tabaci populations had ever been recorded on this island prior to this event, the arrival of a new biotype of B. tabaci was highly suspected. Therefore, the B. tabaci populations of La Réunion were more closely investigated (Chapter 5). Using RAPD-PCR and cytochrome oxidase I (COl) gene sequence comparisons, two genetic types of 5. tabaci were distinguished. One type was assigned to biotype B and the other was genetically dissimilar to the populations described elsewhere and was called Ms after the name of the Mascarenes Archipelago. This new biotype formed a distinct group that is sister to the B biotype group to which also the Q biotype belongs. The Ms biotype is thought to be indigenous to the region as it was also detected on Mauritius, Seychelles, and Madagascar. The divergence from the group formed by the B and Q biotypes was estimated to date back more than 3 ± 0.3 million years ago. Both B and Ms populations of B. tabaci induced silverleaf symptoms on Cucurbita sp., and were able to acquire and transmit TYLCV.The recent colonisation of the B. tabaci biotype B on La Réunion provided a great opportunity to analyse its colonisation process (Chapter 6). As biotypes of B. tabaci are morphologically indistinguishable, a set of 8 microsatellite markers was developed and used to survey the level of genetic variability and the population dynamics of both the indigenous biotype Ms and the invasive biotype B. The results showed that microsatellite analysis, combined with bayesian statistics can efficiently distinguish both biotypes. Genetic diversity within the biotypes Ms and B were comparable, suggesting that the introduction of biotype B was not associated with a severe genetic bottleneck. More than a single introduction might have been responsible for its invasion and these might have occurred several years ago. Interestingly, within a few years the introduced biotype has reached the same level of geographic structuration (Fst) across the island as the local biotype. Biotype B has spread towards all parts of the island, although it has not wiped out the resident biotype in any sector. However, the relative proportions of the two biotypes are far from constant and vary according to geographic or ecological factors. Possibly the distribution of their host plants is of importance here. Indeed the B biotype is dominant on vegetable crops, whereas the Ms biotype is predominant on weeds. Nevertheless, some B populations tend to be genetically more similar to Ms genotypes than other isolated B populations and this is suggestive for some introgression. However, more data are needed (and, perhaps more evolutionary time) to definitely document the presence or absence of hybrids and the ability of both biotypes to interbreed.As a first step towards begomovirus control in cultivated tomatoes, in Chapter 7 a series of accessions of six Lycopersicon species were tested and compared for their potential levels of resistance to TYLCV and to another, Caribbean begomovirus, Potatoyellow mosaic virus (PYMV). PYMV and TYLCV incidence and severity were quantified after vector and graft inoculation in separate experiments. The results obtained indicate that resistance to both viruses may exist in a single genotype. A high level of resistance to both viruses was observed in L chilense LA 1969 after vector inoculation and which was not overcome after graft-inoculation of TYCLV. In two accessions of Lycopersicon pimpinellifolium (INRA-hirsute and La l478) a partial resistance to both viruses was observed after vector inoculation that was overcome after graft inoculation. It was hypothesized that this resistance represented vector resistance to begomovirus transmission. Additionaily, L. pimpinellifolium LA2187-5 was found highly resistant to PYMV but susceptible to TYLCV.After this first screening for begomovirus resistance, the resistance to TYLCV as observed in two wild genotypes of L. pimpinellifolium (INRA-hirsute and WVA 106) was studied into some further detail. TYLCV incidence, symptom severity, and virus content were assessed after massive viruliferous whiteflies inoculation or grafting (Chapter 8). A moderate resistance, which was overcome at high inoculation pressure, was observed for TNRA-hirsute. This partial resistance showed a typical quantitative resistance pattern Based on Chapter 7 plus the graft and vector inoculation results obtained in Chapter 8, the partial resistance in INRA-hirsutc was concluded to act on vector transmission efficiency. This resistance could therefore be effective against other begomoviruses as well.Finally, in Chapter 9 the results obtained in the experimental Chapters are discussed with respect virus evolution, B. tabaci population dynamics, and potential options to control begomovirus disease in tomato.