Sequencing a Strawberry Germplasm Collection Reveals New Viral Genetic Diversity and the Basis for New RT-qPCR Assays

Abstract

Viruses are considered of major importance in strawberry (Fragaria × ananassa Duchesne) production given their negative impact on plant vigor and growth. Strawberry accessions from the National Clonal Germplasm Repository were screened for viruses using high throughput sequencing (HTS). Analyses of sequence information from 45 plants identified multiple variants of 14 known viruses, comprising strawberry mottle virus (SMoV), beet pseudo yellows virus (BPYV), strawberry pallidosis-associated virus (SPaV), tomato ringspot virus (ToRSV), strawberry mild yellow edge virus (SMYEV), strawberry vein banding virus (SVBV), strawberry crinkle virus (SCV), strawberry polerovirus 1 (SPV-1), apple mosaic virus (ApMV), strawberry chlorotic fleck virus (SCFaV), strawberry crinivirus 4 (SCrV-4), strawberry crinivirus 3 (SCrV-3), Fragaria chiloensis latent virus (FClLV) and Fragaria chiloensis cryptic virus (FCCV). Genetic diversity of sequenced virus isolates was investigated via sequence homology analysis, and partial-genome sequences were deposited into GenBank. To confirm the HTS results and expand the detection of strawberry viruses, new reverse transcription quantitative PCR (RT-qPCR) assays were designed for the above-listed viruses. Further in silico and in vitro validation of the new diagnostic assays indicated high efficiency and reliability. Thus, the occurrence of different viruses, including divergent variants, among the strawberries was verified. This is the first viral metagenomic survey in strawberry, additionally, this study describes the design and validation of multiple RT-qPCR assays for strawberry viruses, which represent important detection tools for clean plant programs.

Construction of Infectious cDNA Clone of Brassica Yellows Virus Isolated from Strawberry and Establishment of TaqMan RT-qPCR

“…Disease control relies on accurate viral identification in strawberry production. Common methods for detecting strawberry viruses include ELISA, RT-PCR, and RT-qPCR [ 17 , 19 , 22 ]. In previous studies, multiplex RT-PCR methods have been developed to detect three genotypes of BrYV simultaneously [ 23 ].…”

“…Rapid, specific, and sensitive virus detection methods are essential for the investigation of viruses. Many methods have been used for the detection of strawberry viruses, including reverse-transcription PCR (RT-PCR), enzyme-linked immunosorbent assays (ELISA), and real-time fluorescence quantitative RT-PCR (RT-qPCR) [ 17 , 18 , 19 ]. Among these methods, conventional RT-PCR is known to be less sensitive than real-time RT-qPCR, and ELISA is restricted by the preparation of highly effective antibodies.…”

New Virus Diagnostic Approaches to Ensuring the Ongoing Plant Biosecurity of Aotearoa New Zealand

“…An outbreak of the disease in California in 2002 and 2003 caused an estimated loss of more than USD50 million [ 91 ]. Recently, HTS has been playing a crucial role in unravelling the etiology of several diseases in strawberry [ 92 ], including the identification of SPV1, SCrV-3, and SCrV-4 associated with decline symptoms. SPV1 is a tentative member of the genus Polerovirus in the family Solemoviridae, which was first identified in Canada in 2013 [ 89 ] and subsequently reported in the USA in 2016 [ 93 ] and in the Czech Republic in 2021 [ 94 ].…”

Transmission of Diverse Variants of Strawberry Viruses Is Governed by a Vector Species

“…Nearly 30 different viruses have been reported to infect strawberries, which are predominantly transmitted by different aphid species [ 27 , 28 , 29 , 30 , 31 ]. It is recognized that yield losses are affected by frequently observed mixed viral infections [ 32 , 33 ].…”