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

Naomi Nzilani Mumo,Fredah K Rimberia,Francesca Stomeo,Joyce Njoki Njuguna,Elijah Miinda Ateka,George Ochieng’ Asudi,George Edward Mamati,Laura M Boykin,Roger Pelle,Eunice M Machuka

doi:10.3389/fmicb.2020.00205

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.

Highlights

Papaya (Carica papaya L.) is an important fruit crop both in the tropical and subtropical regions (Mishra et al, 2007)
To provide an insight into viruses associated with papaya ringspot disease (PRSD) symptoms in Kenya, 48 leaf samples were sequenced using the Illumina MiSeq platform
Through Illumina MiSeq sequencing, we characterized complete genome sequences of Moroccan watermelon mosaic virus (MWMV), a Potyvirus; cowpea mild mottle virus (CpMMV), a Carlavirus, and two novel yet divergent Carlaviruses in symptomatic and asymptomatic papaya leaves collected from Kenyan fields

Summary

Introduction

Papaya (Carica papaya L.) is an important fruit crop both in the tropical and subtropical regions (Mishra et al, 2007). The levels of vitamins A and C from one medium-sized papaya (whose edible portion is about 350 g) exceed the Dietary Reference Intakes of 3000 IU for vitamin A and 90 mg for vitamin C, as established by the United States Food and Nutrition Board for adult minimum daily requirements (Ming et al, 2008). Consumption of this fruit is important in preventing vitamin A deficiency, a cause of childhood blindness in many developing countries, including Kenya (Oyunga et al, 2016). The enzyme is purified and used in foods, beverages, pharmaceuticals, and by manufacturing industries (Yogiraj et al, 2014)

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