First Report of Cucumber Green Mottle Mosaic Virus Infecting Zucchini (Cucurbita pepo) and Wax Gourd (Benincasa hispida) in China.

Abstract

Cucumber green mottle mosaic virus (CGMMV) was first discovered in China in 2003 and caused an epidemic in 2005. In China, the virus has been reported in gourd crops including watermelons, cucumbers, melons, etc (Sui et al. 2019). In Shandong Province, China from September 2014 to 2017, approximately 30% of zucchini (Cucurbita pepo) and wax gourd (Benincasa hispida) plants in commercial cucurbit fields, the two most important cash crops, exhibited chlorosis, mosaic, and mottling symptoms suspected to be caused by a tobamovirus. To identify the causative pathogens, ten zucchini and 15 wax gourd samples were collected from the commercial cucurbit fields. Total RNA was extracted and all samples were tested using reverse transcription PCR (RT-PCR) with TobamodF/TobamodR and TobamodF2/TobamodR2 (Li et al. 2018a). Five common Cucurbitaceae viruses were also tested: cucumber mosaic virus, papaya ringspot virus, squash mosaic virus, watermelon mosaic virus, and zucchini yellow mosaic virus (Ali et al. 2012). All samples generated positive results using tobamovirus generic primers but were negative for the five common Cucurbitaceae viruses. Amplification products (880 bp) from all samples were inserted into pMD19-T and recombinant clones were selected for Sanger sequencing. The results showed that zucchini green mottle mosaic virus, CGMMV, and tobacco mosaic virus (TMV) were detected in zucchini samples. CGMMV and TMV were detected in the wax gourd samples. To confirm the presence of these viruses, RT-PCR was performed using specific primer pairs, including CGMMV-cpf/CGMMV-cpr (Chen et al. 2006), ZG-F/ZG-R (Li et al. 2018b), and TMV-CP-F/TMV-CP-R (Srivastava et al. 2015). CGMMV was detected in all samples, with four zucchini and nine wax gourds only containing CGMMV. Zucchini (n=4; CGZ1-CGZ4) and wax gourd (n=4; CGWX1-CGWX4) isolates were cloned into pMD19-T and sequenced bidirectionally. The BLASTn results confirmed the presence of CGMMV, and the sequencing results were processed using DNAMAN Version (Lynnon Biosoft, San Ramon, CA, USA) and submitted to the GenBank database (https://www.ncbi.nlm.nih.gov/). A phylogenetic tree based on the CGMMV coat protein (CP) was constructed using CGZ1-CGZ4 (OP779762-OP779765), CGWX1-CGWX4 (OP779766-OP779769), and representative CGMMV sequences from GenBank. Sequence analysis of the CP demonstrated that CGMMV-zucchini and -wax gourd isolates belonged to an independent branch of the Chinese muskmelon AH-FT197 isolate (KU175639) and had 100% identity with the AH-FT197 isolate. To confirm their infectivity, leaf sap extract of CGZ4 and CGWX4 in phosphate buffer (0.1 M, pH 7.0) was mechanically inoculated on leaves of virus-free zucchini seedlings (Cucurbita pepo cv. Zaoqingyidai, 4-leaf-stage, n = 10) or virus-free wax gourd seedlings (Benincasa hispida cv. Tiezhu 2, n = 10). Ten days after inoculation, all plants exhibited symptoms (systemic chlorosis, mosaic, and mottling) similar to those of diseased plants in the field. Control seedlings inoculated with phosphate buffer remained symptomless. RT-PCR analysis using the CGMMV-cpf/CGMMV-cpr primer confirmed that all ten zucchini or wax gourd seedlings were infected with CGMMV, and all the control plants were free from CGMMV. To the best of our knowledge, this is the first report on zucchini and wax gourd as natural hosts for CGMMV in China. CGMMV is a highly contagious seed-borne virus and further attention should be paid to its spread in cucurbit crops.