Characterization of the virome of shallots affected by the shallot mild yellow stripe disease in France

To elucidate the etiology of a new disease of shallot in France, double-stranded RNAs from asymptomatic and symptomatic shallot plants were analyzed using high-throughput sequencing (HTS). Annotation of contigs, molecular characterization and phylogenetic analyses revealed the presence in symptomatic plants of a virus complex consisting of shallot virus X (ShVX, Allexivirus), shallot latent virus (SLV, Carlavirus) and two novel viruses belonging to the genera Carlavirus and Potyvirus, for which the names of shallot virus S (ShVS) and shallot mild yellow stripe associated virus (SMYSaV), are proposed. Complete or near complete genomic sequences were obtained for all these agents, revealing divergent isolates of ShVX and SLV. Trials to fulfill Koch’s postulates were pursued but failed to reproduce the symptoms on inoculated shallots, even though the plants were proved to be infected by the four viruses detected by HTS. Replanting of bulbs from SMYSaV-inoculated shallot plants resulted in infected plants, showing that the virus can perpetuate the infection over seasons. A survey analyzing 351 shallot samples over a four years period strongly suggests an association of SMYSaV with the disease symptoms. An analysis of SMYSaV diversity indicates the existence of two clusters of isolates, one of which is largely predominant in the field over years.


Introduction
Economically important cultivated Allium species are garlic (Allium sativum), leek 47 (Allium ampeloprasum var. porrum), onion (Allium cepa), and its relative shallot (Allium 48 cepa L. var. aggregatum) [1]. Shallot is mainly cultivated for culinary purposes, while 49 onion and garlic are also used in traditional medicine. Viral infections are a significant 50 problem for all Allium crops, even more so in the case of shallot and garlic which are 51 exclusively vegetative propagated, leading to the accumulation of viruses in planting 52 material [2]. Due to their prevalence and the damages they cause, the most 53 economically important Allium viruses are members of the genus Potyvirus, particularly 54 onion yellow dwarf virus (OYDV) and leek yellow stripe virus (LYSV). In the 55 Mediterranean basin, shallot yellow stripe virus (SYSV) and turnip mosaic virus (TuMV) 56 have also been described infecting Allium species, as well as four other potyviruses of 57 lower incidence, even though TuMV has not been reported on shallot so far [2]. 58 Potyviruses and carlaviruses are transmitted non persistently by aphids and frequently 59 found on cultivated Allium species. The first described Allium-infecting carlavirus was 60 shallot latent virus (SLV, synonym with garlic latent virus, GLV), which seems to be 61 asymptomatic in garlic, onion and shallot when in single infection but can cause 62 significant yield losses in the presence of potyviruses due to synergistic effects [3]. lead to increased damages [2]. Besides the viruses belonging to the Allexivirus, 71 Potyvirus, and Carlavirus genera, five other viruses infecting Allium species have been 72 described, generally with limited incidence, including iris yellow spot virus (IYSV), a 73 member of the genus Orthotospovirus, reported on shallot [4]. 74 In 2012, a new disease was observed in the west of France in shallots. Symptoms 75 consisted of yellow stripes on the leaves, associated with a loss of vigor, considered 76 as moderate as compared to that caused by OYDV or LYSV (Fig 1). This gave its name 77 to the disease, shallot mild yellow stripe disease (SMYSD). Early tests revealed that 78 the new disease could be observed in plants that test negative for OYDV and LYSV, 79 indicating that these two viruses were not involved. In parallel, meristem-tip culture  were recorded using a 0 to 3 notation scale for both leaf striping and loss of vigor. The

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"0" score is defined as no symptom, the "3" score is defined as a symptomatology 99 equivalent to that observed on control plants infected by OYDV. The "1" and "2" scores 100 are used for symptoms of intermediate intensity.  After quality trimming and demultiplexing steps [6], reads were assembled into contigs 110 which were annotated by BlastN and BlastX comparisons [7]  of the host range experiments using the silica-capture procedure 2 described by [8].

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The viruses were detected by two-step RT-PCR assays, following the procedure 121 already described [9] and using specific primers (Table S1). The amplified fragments 122 were visualized on non-denaturing 1% agarose gels and, if needed, submitted to direct 123 Sanger sequencing on both strands (GATC Biotech, Mulhouse, France). shallot virus X (ShVX) 128 The 5' ends of the viral genomes sequences were determined or confirmed 129 using the 5' Rapid Amplification of cDNA Ends (RACE) strategy and internal primers 130 designed from the genomic contigs (Table S1) following the kit supplier's 131 recommendations (Takara Bio Europe/Clontech, Saint Germain-en-Laye, France).

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The 3' ends were amplified using forward internal and polyA-anchored LD primers 133 (Table S1) as described [10]. Internal gaps and regions of low coverage were 134 determined or confirmed by direct sequencing of RT-PCR fragments obtained using 135 internal primers designed from the contigs (Table S1). All amplified fragments were visualized on non-denaturing agarose gels and directly sequenced on both strands by 137 Sanger sequencing (GATC Biotech).       After demultiplexing, quality trimming, and de novo assembly, BlastN and BlastX 184 comparisons of the contigs obtained with the GenBank database showed that all 185 sources but one (13-01) were infected by more than one viral species (Table 1)   In the end, the complete genomic sequences of seven viral isolates were 207 obtained ( genome sequences of three additional ShVX isolates (two from sample 13-01, and one 211 from sample 13-04) and of one additional SLV isolate (from sample 13-03) were also 212 obtained during the assembly process (Table 1) but no specific effort was made to 213 complete their missing 5' and 3' genome ends.

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Besides the whole genome sequence determined for the novel carlavirus,       (Table   297 S2), indicating that they belong to the same species. Remarkably the L28079 298 sequence was described in GenBank database as "shallot potyvirus (probably Onion 299 yellow dwarf virus)" indicating that SMYSaV had been observed previously in shallot 300 in Russia but that its originality and distinctness had not been recognized at the time.

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The four symptomatic samples analyzed by HTS in the present study were all 302 found to be infected by SMYSaV (Table 1) Table 1). The  supported by a high bootstrap value (Fig 4). The phylogenetic analysis based on the 358 CP sequences of allexivirus members and of the available ShVX isolates retrieved 359 from GenBank confirmed this conclusion (Fig S1). Moreover, the sequences reported     (Table 1). 404 Similarly, SMYSaV was detected in 75% of the inoculated shallots and in 100% of the  (Table S1). Twenty-two samples were found to be infected by OYDV or/and 427 LYSV, with a mean of striping score of 2.43 ± 1.03 and a mean score of 1.5 ± 1.46 for 428 the loss of vigor. In the remaining samples, the incidence of SMYSaV was found to be 429 quite high (27.2%) and was highly correlated with the presence of striping symptoms.   The diversity of SMYSaV was also analyzed, using the nucleotide sequence of 456 a short fragment of the CP gene targeted by the RT-PCR diagnostic assay. The average pairwise nucleotide divergence was 1.4% between isolates in this region.

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More interestingly, the diversity could be structured into two distinct clusters, as  SMYSaV infection is strongly associated with striping symptoms, with a severity 506 comparable to those caused by OYDV and/or LYSV infection; on the other hand, the impact of SMYSaV infection in terms of loss of vigor is moderate, as reported for the 508 SMYSD, and quite different from the more severe loss of vigor associated with OYDV 509 and/or LYSV infection (Fig 5). The four symptomatic samples analyzed by HTS were 510 infected with a complex of viruses, which is coherent with the strictly vegetative mode 511 of propagation of shallot. Depending on the sample, various combinations of agents 512 were found, involving SMYSaV and ShVS, ShVX and/or SLV (Table 1)