Garlic (Allium sativum L.) is an economically important crop cultivated worldwide, and commonly used as a popular spice and also for its medicinal value [1]. Asia is the main region for garlic cultivation, with China being the largest producer, contributing more than 75% of the total production in the world [2]. China is an important garlic exporter, and the occurrence of viral diseases seriously affects the yield and quality of garlic [3]. According to the Food and Agriculture Organization of the United Nations (FAO), garlic is grown in about 100 countries and regions around the world [4]. In 2019, China produced the maximum quantity of garlic in the world, with an output of 1.7617 million tons. The occurrence of viral diseases in garlic seriously affects the economy and trade. More than 20 types of viruses are reported to infect garlic worldwide including China. Eight species are currently identified in the genus Nanovirus: Black medic leaf roll virus (BMLRV), Cow vetch latent virus (CvLV), Faba bean necrotic stunt virus (FBNSV), Faba bean necrotic yellows virus (FBNYV), Faba bean yellow leaf virus (FBYLV), Parsley severe stunt associated virus (PSSaV), Pea necrotic yellow dwarf virus (PNYDV), Pea yellow stunt virus (PYSV), Sophora yellow stunt virus (SYSV), Subterranean clover stunt virus (SCSV), and Milk vetch dwarf virus (MDV) [5, 6]. MDV belongs to genus Nanovirus, which usually presents severe stunting, leaf rolling and other symptoms; however, the symptoms differ in different plants [7]. MDV relies primarily on aphids for transmission [8]. Since MDV was detected from pea and broad bean in Japan in 1968, the host range of MDV has been expanding. So far, MDV is known to infect tobacco, pepper, tomato, faba bean, green gram, peas, cowpea, broad bean, and papaya [8]. The genome of the members of the genus Nanovirus consist of eight individually encapsulated circular single-stranded (css) DNA components, each of 0.9 to 1.0 kb [7]. In this study, MDV was isolated from garlic in Shandong Province, China and its whole genome was amplified and sequenced.
Symptoms of dwarfing, yellowing, and reddening of garlic first appeared in early 2019 (Fig. 1A). This was suspected to be caused by MDV, and spread in the garlic cultivation areas around Shandong Province, China. By 2020, the incidence of the disease increased from 2% to 5%. In April 2019 and 2020, twenty symptomatic (exhibiting dwarfing, yellowing, and reddening symptoms) and ten asymptomatic garlic samples, and four aphid samples were collected from the garlic plants growing in the open fields in Taian, Jining, and Jinan City of Shandong Province, China (Fig. 1A). To determine whether the reported symptoms were associated with MDV infection, the total DNA was extracted following the Super Plant Genomic DNA Kit manual (Tiangen Biotech, Co., Ltd., Beijing, China); circular DNA was amplified using the extracted total DNA as template through rolling circle amplification (Phi29 DNA Polymerase, TransGen Biotech) before being digested with restriction enzymes. Specific primers were used to amplify the whole genome of MDV (Suppl. Table S1). Amplified fragments were approximately 1 kb, as determined using agarose gel electrophoresis. MDV was detected in all twenty symptomatic garlic samples and four aphid samples, but was absent in the ten asymptomatic garlic samples. Amplicons from one garlic and one aphid sample were cloned into a pMD18-T vector and assessed by Sanger sequencing (Takara, Shiga, Japan). Twelve cssDNA components consisting of eight MDV genome (DNA-N, DNA-C, DNA-M, DNA-R, DNA-S, DNA-U1, DNA-U2, and DNA-U4) and four alphasatellite DNAs (DNA-C1, DNA-C2, DNA-C3, and DNA-C10) were amplified from MDV garlic and aphid isolates, respectively (Suppl. Table S2).
The phylogenetic trees of all the genes in garlic isolates were clustered with MDV (Fig. 1B, Fig. 2). The identity rate of garlic and aphid isolates and related satellites was 100%. The results of nucleotide and amino acid analysis showed that the five strands (DNA-N, DNA-C, DNA-M, DNA-R, and DNA-S) of garlic isolates from China had the highest similarity with the garlic isolate, but had great variation with other isolates from GenBank database. Three strands (DNA-U1, DNA-U2, and DNA-U4) were amplified from garlic isolates for the first time. DNA-U1 and DNA-U2 remained highly conserved, while DNA-U4 was the least conserved and had the greatest variability. Four alphasatellite DNAs (DNA-C1, DNA-C2, DNA-C3, DNA-C10) remain highly conserved. DNA-C1 had the greatest variation, and had greater variability with the isolates from garlic (Suppl. Table S3). Prior to this study, only part of the MDV sequence was reported in GenBank database. In this study, the whole genome sequence of garlic isolate of MDV was amplified. The study showed that DNA-U4 and DNA-C1 had great variability with the published sequences in GenBank database (Suppl. Table S3, Suppl. Fig. S1). What is the cause of the MDV sequence variation? One possibility is aphid feeding, but the sequence of MDV aphid isolate is consistent with the results of garlic isolate, ruling out this possibility. Further, considering virus recombination, we used RDP4 to perform recombination analysis on MDV isolates, and the results showed that there was a potential recombination event in DNA-R, DNA-U1, and DNA-U2 [9]. We suspect that the mutation may be due to gene recombination and base mutation (Suppl. Fig. S2).
Further analysis indicated that these sequences clustered together with MDV isolates. MDV is transmitted by aphids in a circulative non-propagative manner. Virus-free aphids (approximately 60) were used for virus transmission from MDV positive plants to two garlic leaf seedlings. Twelve garlic plants were equally divided into MDV infected and control groups. One month after inoculation, all the test plants displayed typical symptoms of dwarfing, yellowing, and reddening (Suppl. Fig. S3), whereas plants in the control group showed no symptoms. PCR amplification was performed using specific primers MDV-CPF (ATGGTKRGCAATTGGAATYGGYYTGG)/ MDV-CPR (TTAMAVTTCAATATAHAYTCTRTGTTCTA).
This study confirmed the infection of garlic plants by MDV in China. This is the first report of the whole genome of MDV isolated from garlic. MDV may pose a potential threat to garlic cultivation in China. More efforts are required to prevent further spread of MDV.
Nucleotide sequence accession numbers: The complete genome sequences of isolate and its alphasatellites have been deposited in GenBank under accession numbers OK274036-OK274051, OL962714-OL962721.
