Virome Survey of Banana Plantations and Surrounding Plants in Malawi

Abstract

A virome survey of banana plantations and their surrounding plants was carried out at nation-wide level in Malawi using virion associated nucleic acids (VANA) high throughput sequencing (HTS) on pooled samples and appropriate alien controls. In total, 366 plants were sequenced, and 23 plant virus species were detected, three species on banana (275 plants) and 20 species in surrounding plants (91 plants). Two putative novel virus species; ginger tymo-like virus and pepper derived totivirus were detected and confirmed by RT-PCR on ginger and pepper. Nine known virus species and detected a host plant was identified for two of them. No viral exchange between banana and surrounding plants was observed. Results from the VANA protocol, applied to pooled banana samples, were compared with previous targeted PCR results obtained from individual banana samples. HTS test detected better BanMMV than IC-(RT)-PCR on individual samples (better inclusivity) but detected with much lower sensitivity BBTV and BSV species, often with less than 10 reads per sample. Detection of novel and known viruses and new host plants calls for strengthened sanitory and phytosanitory measures within and beyond banana production systems. Our research confirms that HTS sensitivity depends on sampling, pooling protocol and targeted virus species.

Keywords: detection; high throughput sequencing; new host plant; novel viruses; plant viruses; virome survey.

Figure 1

(a) Survey sites and banana cultivation zones. (b) Collection site for weeds and other plants. In (a) the green dots represent the survey sites and Zone 1 is shaded yellow, Zone 2 brown, Zone 3 dark blue and Zone 4 orange. For (b) green dots sites where samples of other crops were collected and red dots weed collection sites.

Figure 2

Schematic representation of sample pooling for VANA sequencing, the comparison of results with individual targeted test (IC-(RT)-PCR) from Masangwa et al. under review [3], and the independent confirmation of the new detections (new virus, new host and new to Malawi).

Figure 3

Genome organization of the novel virus of the family Tymoviridae. The long grey ORF represents ORF1 (RdRp in frame 1). Positions of the conserved sequence domains in the encoded proteins are indicated by colors: vmethyltransf (Pink color), Salyut (green color), Peptidase C21 (purple color), viral helicase (pale orange color), and the Tymoviridae RdRp (brown color). The ORF2 in dark blue color (coat protein in Frame 2) contains the Tymo-coat (yellow color) conserved domain.

Figure 4

Genome organization of novel virus of the family Totiviridae with ORF1 (Coat protein) in orange and ORF2 (RdRp) in brown. Localisation of the conserved sequence domains in ORF1 and ORF2 are indicated by blue and pink colors, respectively.

Figure 5

Maximum likelihood phylogenetic tree of nearly complete genomes of the new virus identified in this study with other viruses from the Tymoviridae family (green = marafiviruses; black = tymoviruses; blue = maculaviruses; and purple = root).

Figure 6

Maximum likelihood phylogenetic tree of nearly complete genomes of the new Totivirus identified in this study with other viruses from the Totiviridae family (blue = totiviruses; green = victoriviruses, black = Leishmaniavirus; brown = Giardiavirus; and purple = root).

Figure 7

RT-PCR confirmation of (a) novel Tymoviridae and (b) novel Totiviridae detection on individual samples from the HTS pools.