Distribution and Genetic Diversity of Tomato Yellow Leaf Curl Virus, Associated Whitefly Vectors and the Response of Selected Tomato Varieties to the Virus

Abstract

Tomato (Solanum lycopersicum) is an important fruiting vegetable cultivated worldwide because of it’s commercial and high nutritional value. In Kenya, tomato is predominantly cultivated by small scale farmers but its production is constrained biotic constraints such as insect pests and diseases due to bacteria, fungi, nematodes and viruses. Viruses are the third significant constraint to tomato production and this includes viruses in the genus Begomovirus. About 60 begomovirus species affect tomato plants in the tropics and subtropics leading to yield losses of up to 100 %. Begomoviruses are spread by a vector Bemisia tabaci. In Kenya, TYLCV-like virus was reported for the first time in tomato crops in 1996. Since then no comprehensive research has been conducted to establish the genetic diversity of the virus, how commonly grown tomato varieties respond to the virus and the diversity of vectors involved in its transmission. This study aimed at contributing to the development of sustainable management strategies of tomato yellow leaf curl disease in tomato crops in Kenya by determining the genetic diversity of Tomato yellow leaf curl virus present, that of associated vectors and the response of commonly grown tomato varieties to TYLCV. A field survey was carried out in eight major tomato growing regions in Kenya between September and December 2018 and January to March 2019. A total of 259 fields were surveyed and data collected on tomato leaf curl disease prevalence, incidence, and severity. The presence of the virus was further confirmed using DAS –ELISA and molecular techniques. Estimates of whitefly populations colonizing sampled tomato crops was done through direct counting of adult whiteflies on the underneath of five topmost leaves. Adult whitefly samples were collected using a hand held aspirator, carried in 95% ethanol and analyzed using DNA barcoding technique. Screening of selected tomato varieties to tomato leaf curl disease was done at KALRO Mwea in two seasons; September to December 2018 and May to August 2019. The experiments were laid in randomized complete block design with three replications. Each plot had 25 plants and data was collected on TYLCD disease incidence, severity and population of whiteflies from 15 plants per plot. Data on disease incidence, severity, whitefly populations was analyzed using Analysis of Variance (ANOVA) and Pearson correlations using SAS software (version 9.1). Molecular data was analyzed using phylogenetic relationships, recombination analyses and population genetics. This study established that the disease prevalence, incidences and severity varied between the Counties and among fields within a County. There was a significant difference on TYLCD incidences across the Counties and amongst the sampled varieties (p≤0.05). Kwale County had the highest disease incidence while Bungoma had the least. The disease incidence was generally lower in hybrids compared to conventional varieties. During screening for the response of selected varieties to the disease, it was observed that all tomato varieties tested were susceptible to the disease and there was no significant difference in disease incidence and severity between the test varieties in both season 1 and 2. Moreover, whitefly populations were not statistically different in both seasons and there was no correlation between whitefly population and TYLCD incidence and severity. However, the whitefly population differed significantly (p≤0.05) on the test varieties. Serological assays confirmed the presence of Tomato yellow leaf curl virus but further analysis using metagenomics revealed that the begomovirus symptoms observed on tomato crops are caused by Tomato leaf curl Arusha virus (ToLCArV). Twelve complete genomes were obtained from the samples with an average coverage of 99.9%. The sequences showed 95.7-100% identity amongst themselves. Analysis of amino acid sequences showed the highest identities in the regions coding for the coat protein gene (98.5–100%) within the isolates, and 97.1–100% identity with the C4 gene of ToLCArV. Phylogenetic algorithms clustered all Kenyan isolates in the same clade with ToLCArV, confirming the isolates to be a variant of the Tanzanian virus. There was no evidence of recombination within the isolates. Estimation of selection pressure within the virus population revealed the occurrence of negative or purifying selection in five out of the six coding regions of the sequences. Though begomoviruses are vectored by Bemisia tabaci, all the 163 whitefly samples collected from tomato plants and analysed in this study were Trialeurodes vaporariorum species and had no intra population diversity. Demographic analysis indicate population expansion of T. vaporariorum observed. It is therefore concluded that begomovirus symptoms found in tomato plants in Kenya are caused by Tomato leaf curl Arusha virus. Breeding programs should consider developing cultivars resistant to this virus. However, there is need to evaluate the role of the complex agroecosystems in tomato fields in the transmission of ToLCArV. Further research should be done to determine if the virus is seed transmitted. The information generated in this study will be useful in developing sustainable management strategies of the disease in the country.