Metagenomic Analysis of Plant Viruses Associated With Papaya Ringspot Disease in Carica papaya L. in Kenya

Abstract

Carica papaya L. is an important fruit crop grown by small-and large-scale farmers in Kenya for local and export markets. However, its production is constrained by papaya ringspot disease (PRSD). The disease is believed to be caused by papaya ringspot virus (PRSV). Previous attempts to detect PRSV in papaya plants showing PRSD symptoms, using enzyme-linked immunosorbent assay (ELISA) and reverse transcriptase-polymerase chain reaction (RT-PCR) procedures with primers specific to PRSV, have not yielded conclusive results. Therefore, the nature of viruses responsible for PRSD was elucidated in papaya leaves collected from 22 counties through Illumina MiSeq next-generation sequencing (NGS) and validated by RT-PCR and Sanger sequencing. Viruses were detected in 38 out of the 48 leaf samples sequenced. Sequence analysis revealed the presence of four viruses: a Potyvirus named Moroccan watermelon mosaic virus (MWMV) and three viruses belonging to the genus Carlavirus. The Carlaviruses include cowpea mild mottle virus (CpMMV) and two putative Carlaviruses-closely related but distinct from cucumber vein-clearing virus (CuVCV) with amino acid and nucleotide sequence identities of 75.7-78.1 and 63.6-67.6%, respectively, in the coat protein genes. In reference to typical symptoms observed in the infected plants, the two putative Carlaviruses were named papaya mottle-associated virus (PaMV) and papaya mild mottle-associated virus (PaMMV). Surprisingly, and in contrast to previous studies conducted in other parts of world, PRSV was not detected. The majority of the viruses were detected as single viral infections, while a few were found to be infecting alongside another virus (for example, MWMV and PaMV). Furthermore, the NGS and RT-PCR analysis identified MWMV as being strongly associated with ringspot symptoms in infected papaya fruits. This study has provided the first complete genome sequences of these viruses isolated from papaya in Kenya, together with primers for their detection-thus proving to be an important step towards the design of long-term, sustainable disease management strategies.

Current Developments and Challenges in Plant Viral Diagnostics: A Systematic Review

“…Using Illumina MiSeq sequencing, Mumo and colleagues marked the genome sequences of four different viruses, a potyvirus (Moroccan watermelon mosaic virus) and carlavirus , including the Cowpea mild mottle virus (CpMMV) and two other putative carlaviruses related to Cucumber vein-clearing virus (CuVCV) from papaya leaves in Kenyan fields [ 137 ]. This study is the first report demonstrating the presence of these viruses in papaya in Kenya.…”

Detection of Plant Viruses and Disease Management: Relevance of Genetic Diversity and Evolution

“…HTS allowed elucidating the elusive etiology of some diseases ( Vives et al., 2013 ; He et al., 2015 ), but often it is not possible to find a direct association between the disease and a particular virus ( Tomašechová et al., 2020 ) among those detected in the infected plant. In this case, the diagnostic must be established by fulfilling Koch’s postulates, or at least by finding a tight association between the disease and the presence of a certain virus in field surveys ( Mumo et al., 2020 ).…”

Use of High-Throughput Sequencing and Two RNA Input Methods to Identify Viruses Infecting Tomato Crops

“…One of the main methodological decisions for pool sequencing is the balance between the number of samples to be pooled and the sequencing depth required to detect all viruses present in the pool. According to the literature, the pool composition of different metagenomic studies for plant viromes varied from four to 50 individual samples per pool [ 9 , 12 , 35 , 36 , 37 , 38 , 39 ]. Skums et al [ 40 ] provided a mathematical approach for a pooling strategy for the massive sequencing of human viruses, since the complex nature of these samples imposed restrictions on the pool design [ 40 ].…”