First report of Leek yellow stripe virus, Onion yellow dwarf virus, and the putative allium polerovirus A in elephant garlic (Allium ampeloprasum) in Brazil.

Abstract

Five elephant garlic plants (Allium ampeloprasum L.) showing leaf symptoms of chlorotic streaks and mosaic (Figure 1A and B) were collected, in September 2021, in an experimental area in municipality of Rio do Sul (27°11'07"S, 49°39'39"W), State of Santa Catarina, Brazil. Total RNA was extracted using TRIzol® reagent (Invitrogen, USA), according to the manufacturer's instructions to investigate viral infection. The RNA from all five plants were pooled into a single sample for cDNA library construction with the TruSeq Stranded Total RNA with Ribo-Zero Plant (Illumina) kit, which was then sequenced on the Illumina HiSeq2500 platform (Proteimax Biotechnology LTDA). After high throughput sequencing (HTS), 49 million raw reads (each 151nt) were generated. They were trimmed with the BBduk tool and de novo assembled with the Tadpole assembler tool (Geneious Software version 2022). A total of 28,345 contigs were generated and searched against the NCBI virus genome database using BLASTn and BLASTx, with positive results for two potyviruses, leek yellow stripe virus (LYSV), onion yellow dwarf virus (OYDV), and the putative polerovirus allium polerovirus A (APVA). The trimmed reads were mapped with the BBmap tool (Bushell 2014), using reference sequences for LYSV (NC_004011), OYDV (NC_005029), and APVA isolate Won (MH898527). A total of 806,060 reads were mapped, resulting in the nearly complete genome of LYSV (isolate RDS22-2, 10,268 bp, ON565071), which shared the highest (89.41%) nucleotide (nt) identity with LYSV isolate MG (KP258216). The nearly complete genome of OYDV (isolate RDS22-1, 10,519 bp, ON565070) was assembled using 311,467 reads, being 90.21% nt identical to OYDV isolate G-118 (KF632714). The APVA genome (isolate RDS22-3, 4,367 bp, ON565072, Figure 1C) was assembled from 116,303 reads and it shared the highest (90.73%) nt identity with APVA isolate Won. Subsequently, each sample was RT-PCR screened separately for potyviruses and poleroviruses, using the generic primer pairs NIb2F/NIb3R (Zheng et al., 2010) and Pol-G-F/Pol-G-R (Knierim et al., 2010), respectively. Amplified DNA fragments with approximately 350 bp and 1000 bp were obtained for potyviruses and poleroviruses, respectively, and were sent for Sanger sequencing (ACTGene, Alvorada, Brazil). The Sanger derived partial sequences shared 98 to 100% nt identities with corresponding HTS-derived sequences. The most common virus was LYSV, which was found in three of the five tested samples, whereas OYDV and APVA were only found in one sample each. The plants were also screened with specific primers for each virus, and none of the samples revealed mixed infections. Elephant garlic is primarily utilized for industrial garlic production in several countries, and it is now being researched in Brazil for the same purpose. It can be observed from this study that elephant garlic is susceptible to two of the most common viruses in garlic (LYSV and OYDV), which must be considered in the future while developing resistant varieties or in using thermotherapy and shoot tip/meristem culture to recover virus-free cultivars. LYSV and OYDV have already been described in Brazil infecting Allium sativum (Kitajima 2020). The only complete APVA sequence available is from China (Isolate Won), but no further characterization of the virus has been performed and published. The occurrence of this virus in Brazil highlights the importance of further research to obtain a more robust virus characterization.