First report of watermelon crinkle leaf associated virus-1 (WCLaV-1) in watermelon (Citrullus lanatus) in Australia

Surveys of key cucurbit production regions in New South Wales, Australia were undertaken to catalogue the diversity of viral pathogens affecting field grown cropping systems. Analysis of the collected isolates confirmed a detection of watermelon crinkle leaf associated virus-1 (WCLaV-1) in watermelon constituting the first report of WCLaV-1 in Australia. A mixed infection of WCLaV-1 and watermelon mosaic virus (WMV) was also detected constituting the first report of this type of pathogen combination.

Cucurbits are important summer crops grown across vegetable production regions of Australia, however viral diseases remain a persistent drawback in cucurbit production (Sharma et al. 2016). Between September 2018 and November 2021 surveys were conducted on field grown cucurbit crops across New South Wales (NSW), Australia to identify the resident viral pathogen populations.
Symptomatic field grown watermelon (Citrullus lanatus L.) leaves and fruits were sampled in the Sunraysia region of southern NSW. The field crops displayed virus-like symptoms such as mottle, slight puckering and curling of the leaves, dark green lines and circular lesions on fruits at an incidence of < 5% (Fig. 1a). Total RNA was extracted from leaves of one of the symptomatic watermelon plants using the RNeasy Plant Mini Kit (Qiagen, Germantown, Maryland, USA) and RNA quality control was conducted using the Qubit® Flurometer 2.0 (Invitrogen, Waltham, S. Mulholland Shannon.mulholland@dpi.nsw.gov.au 1 The HTS results were verified by RT-PCR using the protocol outlined in Xin et al. (2017). Briefly, the primers MP-1 F/MP-1R targeted a 1005 bp region of the movement protein (MP), NP-1 F/NP-1R targeted a 902 bp region of the nucleocapsid protein (NP). The RT-PCR was conducted using Invitrogen SuperScript™ III One-Step RT-PCR System (Invitrogen, Waltham, Massachusetts, USA). RT-PCR products were purified using the Bioline Isolate II PCR and Gel Kit™ (Meridian Bioscience, London, UK) and sent for sequencing at the Australian Genome Research Facility Ltd. The sequences were analyzed using Geneious Prime 2020 (Kearse et al. 2012). RT-PCR verification of the HTS results produced a movement protein gene (MP-1 F/R primers) PCR product that matched WCLaV-1 GenBank accession KY781185.1 with 99% nt. The single WCLaV-1 accession match for the nucleocapsid protein gene (NP-1 F/R primers) for each sample was KY781186.1 (99% similarity), both of which correlate with the RNA 1 segment of the WCLaV-1 genome.
Subsequent sampling of symptomatic field grown watermelon leaves from a further three plants from the infected property were tested via RT-PCR using the protocol outlined above and returned positive results of WCLaV-1 in a further three samples (Fig. 1b). No further mixed infections of WMV and WCLaV-1 were detected.
The potential for mechanical transmission of WCLaV-1 was first raised by Xin et al. 2017. To further explore this possibility and fulfil Koch's postulates for WCLaV-1, host response inoculation was conducted using watermelon seed 'Sugar Baby' and 'Candy Red'. The cultivars used in the bioassay were selected as they were commercially available at the time of the experiment although no information was available for these varieties regarding their susceptibility or tolerance to WCLaV − 1. A total of 10 plants from each variety were sown into moist coco peat bags and maintained within an insect proof greenhouse on automatic fertigation. Symptomatic watermelon leaf tissue was sourced from the infected field crop (from the second round of sampling) and confirmed to be positive for WCLaV-1 and free of WMV. This leaf tissue was homogenised in a mortar and pestle with 0.1 M phosphate buffer, pH 7.4, at a ratio of 1:5 weight/ volume to prepare the viral inoculum. Once the watermelon seedlings had produced two true leaves, the leaves were dusted with finely ground diatomaceous earth powder, and the WCLaV-1 viral inoculum was gently rubbed onto the leaves. Three control plants of each variety were mock inoculated with healthy watermelon leaf sap (confirmed free of WCLaV-1) to serve as negative controls. The inoculated plants were sampled four weeks later and tested via RT-PCR to confirm the WCLaV-1 infection.
In the greenhouse bioassay, WCLaV-1 infection was confirmed in three of the ten 'Sugar Baby' plants analyzed. However, WCLaV-1 was not detected in any of the 'Candy Red' plants or the mock inoculated plants. The WCLaV-1 infected plants revealed subtle symptoms including leaf bubbling on new growth, leaf distortion near the petiole and vein breaking. Considering the limited literature published on this virus further investigations are necessary to confirm the potential tolerance of some commercial watermelon cultivars to the virus. Additional work on the mechanical transmission efficacy is also required. This is the first report of WCLaV-1 in Australia and the first report of a mixed infection of WCLaV-1 and WMV within a watermelon host. This finding expands the known global geographic range of WCLaV-1. After being first reported in China in 2017 (Xin et al. 2017), recent detections have also been reported in the USA Hendricks et al. 2021) and Brazil (Maeda et al. 2021). With no entry pathway identified for Australia or insect vector identified at present, this may indicate a potential seed-borne transmission pathway. In light of this speculation, it is recommended to screen plants for the presence of WCLaV-1 before carrying out mechanical transmission or cultivar resistance experiments. This was not performed in the current study as the likelihood of seed borne transmission was not well understood at the time of the bioassay, however this hypothesis has now been strengthened with the additional global detections. More work is required to validate this supposition and also to confirm if other cucurbit species or alternate hosts may be affected by this virus.