Molecular characterization and incidence of viruses affecting water melon and pumpkin production in Uganda.

Abstract

Watermelons and pumpkins are cultivated in Uganda for their leaves, fruits, and seeds, thereby contributing to food, nutrition and income security. However, there is limited research and information on constraints and especially viruses affecting their production. The use of resistant cultivars remains the most reliable and environmentally friendly method to increase the annual yields. This study assessed the constraints affecting production, molecular characterization of viruses infecting the crops, and analysis of local cultivars for resistance to Moroccan watermelon mosaic virus (MWMV). Two surveys were conducted in 9 sub-regions namely Acholi, central, east central, Elgon, Lango, south western, Teso, west Nile and western sub-regions based on the importance and availability of watermelon and pumpkin fields.

The first survey was conducted in between June and November 2020 using a structured questionnaire administered to farmers on different management practices, strategies, and current production constraints. During the survey, symptomatic and symptomatic watermelon and pumpkin leaf samples were preferentially selected for virus genome characterization. Descriptive statistics including one-tailed t-test, and chi-square were used for analysis of constraints affecting production. Pests, diseases, drought, high transport and labour costs were the most important constraints in that order. Bacterial wilt, downy mildews, anthracnose, powdery mildews and virus diseases, in this order, were important disease constraints. While white-fly, aphids, melon fly and cut worm were important pests. Judicious application agrochemicals were observed in watermelon fields with possible overreaching effects on the environment.

For virus identification and genome characterization, a total of 90 samples representing 75 (60 symptomatic and 15 asymptomatic) pumpkin and 15 (12 symptomatic and symptomatic) watermelon leaf samples were collected. The samples were pooled in 5 groups for pumpkin and one for watermelon, each containing 15 leaf samples and sequenced using Illumina Hiseq sequencing platform at the Agricultural Research Council Biotechnology Platform, Pretoria. The raw reads were assembled using Genome Detective 1.132. For the first time, six viruses were identified in Uganda infecting pumpkin: (i) Pepo aphid-borne yellows virus (PABYV) (ii) Cucurbit yellow stunting disorder virus (CYSD), (iii) Tomato leaf curl virus, (iv) Tobacco streak virus (TSV), (v) Pepper leaf curl virus, and (vi) Moroccan watermelon mosaic virus (MWMV). One MWMV isolate is a recombinant with major parents from Kenya and minor parent from this study. Complete genome sequences of MWMV and TSV infecting pumpkin in Uganda were assembled and characterized. Primers were designed from next generation sequencing (NGS) data targeting the cylindrical inclusion (CI) and coat protein (CP) of MWMV, PABYV and TSV to confirm presence of the virus in individual samples pooled. In one pooled watermelon sample, (symptomatic and asymptomatic) two complete genomes of PABYV and MWMV were assembled while partial virus genome sequences of Watermelon crinkle leaf- associated virus, Citrullus lanatus cryptic virus, and Cucumis melo cryptic virus were identified.

The second survey was conducted during the June to July season 2022 to document the distribution of MWMV, and PABYV in 9 sub-regions and determine any co-infections in watermelon and pumpkin. These two viruses were detected in all the sub-regions during the first survey. A total of 101 samples were analysed using reverse transcription polymerase chain reaction (RT-PCR) of which, 84.2% of all the samples collected were symptomatic and had an average disease severity of 2.5 on a visual scale of 0-4. Disease incidence in watermelon was highest in east central compared to central sub-region. While in pumpkins, virus-like diseases were observed in all the sub-regions except Teso and Acholi. The incidence of PABYV was 17.8% in all the sub-regions while that of MWMV was 37.0% and four (two of watermelon and pumpkin respectively) samples were co- infected by the viruses.

To increase on the resistance base, an experimental evaluation of the level of resistance of local cucurbits (watermelon, pumpkin, cucumber and loofah) to MWMV was conducted.

This was the most widely occurring virus infecting cucurbits in Uganda. The source of virus inoculum was a pumpkin plant from the field at the National Crops Resources Research Institute (NaCRRI). Local and improved cucurbit cultivars were raised in a screen house in a completely randomized design and inoculated with the MWMV isolate. The reactions to the virus inoculum were recorded for four weeks after inoculation. The severity was evaluated on a scale of 0 to 4. Six cucurbit cultivars (Sugar sweet orange, Sunfish, Oziga yellow, sweety pumpkin, Wujju hard cover, and loofah) all showed a high average virus disease severity of 3, while Zebra-jubilee (watermelon), and Sweat cream, and Oziga simlaw (pumpkin), showed low average virus disease severity. These three cultivars are potential sources of tolerance or partial resistance after confirmation by other methods