Sequencing and variant detection of eight abundant plant-infecting tobamoviruses across Southern California wastewater

Abstract

Tobamoviruses are agriculturally-relevant viruses that cause crop losses and have infected plants in many regions of the world. These viruses are frequently found in municipal wastewater - likely coming from human diet and industrial waste across wastewater catchment areas. As part of a large wastewater-based epidemiology study across Southern California, we analyzed RNA sequence data from 275 influent wastewater samples obtained from eight wastewater treatment plants with a catchment area of approximately 16 million people from July 2020 - August 2021. We assembled 1,083 high-quality genomes, enumerated viral sequencing reads, and detected thousands of single nucleotide variants from eight common tobamoviruses: Bell Pepper Mottle Virus, Cucumber Green Mottle Mosaic Virus, Pepper Mild Mottle Virus, Tobacco Mild Green Mosaic Virus, Tomato Brown Rugose Fruit Virus, Tomato Mosaic Virus, Tomato Mottle Mosaic Virus, and Tropical Soda Apple Mosaic Virus. We show that single nucleotide variants had amino acid-altering consequences along with synonymous mutations, which represents potential evolution with functional consequences in genomes of these viruses. Our study shows the importance of wastewater sequencing to monitor the genomic diversity of these plant-infecting viruses, and we suggest that our data could be used to continue tracking the genomic variability of such pathogens.

Tomato brown rugose fruit virus Mo gene is a novel microbial source tracking marker

“…Notably, the relative abundance of ToBRFV was consistently greater than PMMoV, which is a well established microbial source tracking marker and known to be highly abundant in wastewater (8). This is consistent with reports from studies carried out prior to (44), and in parallel with (29, 45) ours, that also show that ToBRFV is a highly prevalent virus in wastewater.…”

“…New ToBRFV genomes became available on public databases within the last six months. An additional 113 (total 183) from NCBI (28) and 250 assembled ToBRFV genomes from a study of wastewater from Southern California (29) were downloaded (Table 1).…”

Metagenomic analysis of plant viruses in tropical fresh and wastewater

“…Even with this approach, tobamovirus diversity was higher in our study than in other published reports. Rothman and Whiteson (2022) reported the presence of eight tobamoviruses in wastewater (California, USA), while Bačnik with regards to other studies may be due to factors such as the type of sample processing (filtering, concentration, etc. ), library preparation (type of library and amplicon size), sequencing and bioinformatic analysis (Haramoto et al, 2018;Lindgreen et al, 2016).…”

“…As mentioned above, the presence of tobamoviruses in wastewater has previously been described (Jezewska et al, 2018). However, relatively few metagenomic studies have focused on the diversity of plant viruses in wastewater (Bačnik et al, 2020;Rothman & Whiteson, 2022) or river water (Adriaenssens et al, 2018;Solomon & Hewson, 2022). Moreover, we took a conservative approach for viruses' classification, where we considered that viruses belonging to a species were correctly assigned if it was detected by two methods to reduce the probability of false positives or misassignment of reads that could affect our species richness estimation.…”

“…Plant viruses such as tobacco necrosis virus (TNV) and tobacco mosaic virus (TMV) have been reported in sewage since 1960 (Yarwood, 1960). However, with the advent of new and more sophisticated techniques-electronic microscopy, PCR, serology, metagenomics-plant viruses have been detected in a wide variety of water bodies: canals, rivers and streams, ponds, lakes, oceans, and wastewater (Adriaenssens et al, 2018;Mehle et al, 2018;Rothman & Whiteson, 2022). Plant viruses can be transmitted in various ways.…”