Molecular identification and sequence analysis of the pathogen causing Kiwano melon (Cucumis metuliferus) virus disease in Fujian

Abstract:【Objective】Kiwano melon (Cucumis metuliferus) is popular among consumers at home and abroad because of its new exotic shape and unique taste. In recent years, the promotion and planting ar- ea of Kiwano melon in China increased rapidly, and the harm of virus disease increased year by year. The purpose of this study was to provide a theoretical basis for the identification of the types of virus diseases in the planting base of Kiwano melon in Sanming city, Fujian Province, and for the effective prevention and control of virus diseases.【Methods】In this study, the pericarp of Kiwano melon was used as experimental material. Reverse transcription- polymerase chain reaction (RT-PCR) was used to identify the infection status of seven common Cucurbitaceae viruses in Kiwano melon, including Zuc- chini yellow mosaic virus (ZYMV), Watermelon mosaic virus (WMV), Pumpkin mosaic virus (SqMV),Papaya ringspot virus (PRSV), Cucumber mosaic virus (CMV), Turnip mosaic virus (TuMV) and Cu- cumber green mottle mosaic virus (CGMMV). We selected six tested samples and used disposable surgi- cal blade to cut the pericarp with lesions, each sample 0.2 g. After quick freezing in liquid nitrogen, they were stored in -80 °C ultra-low temperature refrigerator. Total RNA was extracted from the peri- carp of Kiwano melon by TIANGEN RNAprpe Pure Plant Kit (Polysaccharides & Polyphenolics-rich). The OD value of total RNA was determined by Thermo. For qualified total RNA, the first strand cDNA was synthesized by the TransGen (TransScript® II One-Step gDNA Removal and cDNA Synthesis Su- perMix) kit. The synthesized cDNA was diluted five times as the template for subsequent PCR. Accord-ing to the corresponding coat protein gene sequence published in NCBI, the virus specific primers were designed in the conserved region of the gene by multiple sequence alignment. ZYMVCP-F/ZYMVCP- R primers were designed at the head and tail of ZYMV coat protein gene to amplify the full length of ZYMV coat protein gene. The PCR reaction system was as follows: 2×Premix Taq 12.5 μL, upstream and downstream primers (10 μmol · L-1) 1 μL, cDNA template 1 μL, ddH2O added to 25 μL. The PCR procedure was as follows: pre denaturation at 94 °C for 3 min, denaturation at 94 °C for 30 s, annealing temperature set according to Tm value of primer, extension time at 72 °C for 60 s, and extension at 72 °C for 5 min after 36 cycles. And then, PCR products were detected by Electrophoresis with 1% agarose gel. The coat protein gene sequences of ZYMV from different countries, regions and plants were downloaded from NCBI, and the evolutionary tree was constructed by MEGA-X software.【Results】The specific bands of ZYMV (284 bp) and WMV (485 bp) were obtained from all six samples. The ob- tained bands of ZYMV and WMV were cut and recovered respectively, and then TA cloning was car- ried out with PMD 18-T (TaKaRa) as vector. After sequencing, it was found that the amplified band length of ZYMV was consistent with the expectation, but the band length of WMV was 39 bp less than the expectation. The results showed that the six samples were only infected with ZYMV. The full length of coat protein gene of ZYMV was obtained from six samples by the designed specific primers ZYM- VCP- F/ZYMVCP- R, and the sequence matching degree between the obtained coat protein gene of ZYMV and that of WMV was 100%, which further proved that the band amplified by WMV primers belonged to the coat protein gene of ZYMV. The results showed that six samples were only infected with ZYMV. ZYMV and WMV are both Potyvirus. The sequence similarity of their coat protein gene was about 64%, and the protein sequence similarity was about 68%. Therefore, it is difficult to distinguish ZYMV and WMV by designing primers in the conserved region of coat protein gene. This would be the reason why WMV primers could amplify bands. PCR combined with gene sequencing could effectively distinguish similar viruses. Phylogenetic analysis showed that ZYMV was widely distributed; 61 iso- lates could be divided into six groups, and there were some regional differences in the distribution of different genotypes. The coat gene of ZYMV (MZ271862) isolate from Fujian Province was located in the branch of ZYMV-II, which was dominated by the domestic isolate. The isolate from Guangxi Siraitia grosvenorii (AJ889244_ Guangxi_Siraitia grosvenorii) was the closest relative. The protein sequence similarity was 99.28%, and only two amino acids were different. Therefore, ZYMV of Fujian Kiwano melon might be transmitted from Guangxi Province.【Conclusion】Generally, fruits are better preserved than leaves, so it is of great significance to use the peel as the test material to study the virus infection. The tested samples were only infected with ZYMV. The infection of Kiwano melon virus dis- ease in Fujian Province was studied for the first time, and the evolutionary origin of ZYMV of Kiwano melon in Fujian Province was clarified, which could provide theoretical basis for further study on the distribution and effective prevention and control of Kiwano melon virus disease.