Molecular characterization of strawberry vein banding virus from China and the development of loop‑mediated isothermal amplification assays for their detection

Strawberry vein banding virus (SVBV) is one of the serious viral pathogens infecting strawberry worldwide. To understand the molecular characterization of SVBV from China, complete genome sequences of sixteen SVBV isolates were cloned and sequenced. Sequence comparison showed they shared high nucleotide sequence identity (93.6–99.5%) with isolates from China and Japan (96.6–98.4%), while relatively low identity with the isolates from Canada (91.9–93.7%) and USA (85.5–85.9%). Phylogenetic analyses based on the complete genome sequence or coat protein (CP) gene showed the SVBV isolates clustered into three clades correlated with geographic distribution. Recombination analyses identified 13 recombinants and 21 recombinant events, indicating frequent and multiple recombinations in SVBV evolution. Furthermore, a sensitive loop-mediated isothermal amplification (LAMP) method was developed for rapid detection of SVBV isolates, which could be especially suitable for seedling propagation, virus-free culture and routine diagnostics in field investigation. This study offers new understanding of the molecular evolution and may help to improve the management of SVBV.

www.nature.com/scientificreports/ 30-60 min, the batch amplification of the target sequence can be realized. The additional advantage of LAMP technique is that final results are directly visible to naked eyes or by using gel electrophoresis. Although LAMP has been successfully applied for the detection of various plant pathogens, no attempt has yet been made to detect SVBV to our knowledge. As strawberry production expands, China now has the largest acreage planted in the world and some of the strawberry viruses and diseases have also emerged 20 . SVBV is widely distributed in the strawberry producing area of China, but the molecular characterization of SVBV in China remains poorly understood. In this study, we cloned and sequenced the complete genome of sixteen SVBV isolates from China and analyzed its genomic characterization. Moreover, we developed and optimized a very sensitive LAMP assay for SVBV diagnostics. This research will be helpful to the investigation and study of virus disease, thus providing theoretical guidance for sustainable strawberry production.

SVBV was prevalent in strawberry plants.
In the field survey, strawberry plants showed the typical symptoms of viral disease, such as appearing stunted, clustered, deformed, and suffering from mosaic, but some plants were symptomless (Fig. 1). To clarify the occurrence of SVBV in strawberry plants, 259 strawberry samples from different regions of China were collected and detected by PCR and sequenced using specific primers. The types of fields that were surveyed including organic production, soil cultivation, substrate culture, elevated cultivation and integrated production. The results showed that 71 samples (27.4%) were SVBV-positive and this virus disease happened very commonly on strawberry in China.
Complete genome sequence characterization and phylogenetic analyses of SVBV. The complete genome sequences of sixteen SVBV isolates from China were cloned and submitted to the GenBank database (Accession Nos: MN956520, MT012732, MT012734, MT027006, MT027007, MT036053, MT036054, MT036055, MT036056, MT036057, KX249738, KX249737, KX249736, KX249735, MF197916 and KT250632). The length of these isolates varied from 7846 to 7942 nts and coding capacity analyses showed the doublestranded DNA genome contained seven putative open reading frames (ORFs), which was consistent with other reported SVBV isolates and other members in the genus Caulimovirus 21 (Supplementary Fig. 1). ORF I encoded the putative viral movement protein involved in cell-to-cell movement of 329 aa with a predicted molecular mass of 37.8 kDa. The conserved DXR motif which may be functionally important was also present. ORF II encoded a putative aphid-transmission-associated protein of 162 aa and 18.5 kDa. ORF III encoded a putative virionassociated protein of 116 aa or 117 aa in some isolates from Beijing with the acc. no. MT036053-MT036055, KT250632 and MT027007. ORF IV encoded a coat protein of 471 aa and 55.0 kDa which contains the conserved zinc-finger domain with the arrangement Cx2Cx4Hx4C typical of all the caulimoviruses. ORF V encoded a putative reverse transcriptase of 704 aa and 80.6 kDa with the motifs of this multifunctional proteins in SVBV: a Leu-zip motif near the N-terminus, an Asp-proteinase domain, a reverse transcriptase domain and an RNase-H domain. ORF VI encoded a putative inclusion body matrix protein of 520 aa and 5.9 kDa. ORF VII encoded a putative protein of 107 aa. The non-coding region (NCR) was between the ORF VI and ORF VII, containing a CAT-like element (GGC CAT ), an eukaryotic promoter TATA box (TAT ATA A) and a poly (A) signal (AAT AAA ).   Fig. 2). At the ORF level, all the isolates from China shared 95.4-99.8% and 95.7-99.8% sequence identity at nt and aa level respectively except for the isolate (HE681085). To elucidate the relationship of different SVBV isolates, a phylogenetic tree was constructed with the available SVBV complete genomic sequences. The phylogenetic tree illustrated that the isolates from USA, the isolates from Canada and the isolates from China and Japan clustered separately into three clades (Fig. 2), which was also in accordance with the sequence comparison. To further confirm the phylogenetic relationship, the coat protein (CP) gene nucleotide sequence based phylogenetic tree was also constructed (Fig. 3). Many Canadian isolates (shown in Fig. 3) were only sequenced for the CP gene and the complete genomic sequences were still unknown. So there are so many variants from Canada compared to Fig. 2. The same topology was observed, indicating that the SVBV evolution was strongly associated with geographical distribution.

High recombinant frequency and multiple recombinant events in SVBV.
A total of thirteen isolates were identified to be potential recombinants with 21 recombination events detected, indicating a relative high recombinant frequency in SVBV. One recombination event was detected in the isolates from China (MT036056, MT731326, KR080547, MT012734 and KP311681) and Japan (LC315804). Two recombination events were detected in the isolates HE681085, MT027006, KX950836, KT250632, MT036055 and MT027007 in different regions while in the isolate MT036055 the two recombination regions overlapped. Three recombination events were detected in the isolate KX787430. The recombination regions were distributed nearly along the whole genome, indicating no recombination hotspot (Supplementary Table S1). Recombination is an important mechanism in virus evolution that can lead to increased or decreased variation and is a major player in virus speciation events leading to emerging viruses. This phenomenon has been reported in single-stranded DNA viruses involving those of begomoviruses and mastreviruses in the family Geminiviridae including geminiviruses and double-stranded DNA virus including cauliflower mosaic virus in the genus Caulimovirus 21 .
Development and optimization of the LAMP assay. LAMP assay conditions were optimized in a stepwise manner, with one parameter modified at a time following the order of gels presented in Fig  www.nature.com/scientificreports/ were formed, while a clear waterfall strip could be formed with higher concentrations. The primer F3/B3 had little effect on the formation of waterfall bands, the same is true for Mg 2+ and Betaine. The optimized amplification was achieved by applying incubation for 45 min at 62 °C. The finally optimized reaction system was 1.2 μM SVBV-FIP/BIP, 0.1 μM SVBV-F3/B3, 2 mM Mg 2+ , 1.6 mM dNTPs and 1.0 M Betaine (Fig. 4). The full-length gels are presented in Supplementary Fig. S4.
Specificity, sensitivity and field applicability of the LAMP assay. The specificity of the LAMP detecting SVBV were confirmed by both the gel electrophoresis and visualized analysis to amplify only DNA from SVBV, with no amplification of the negative control and other viruses (Fig. 5). Obvious bands were not achieved when the dilution multiple exceeded 100 for traditional PCR method, while the LAMP method was 1000 times more sensitive than PCR with the dilution limit of 10 -5 in comparison (Fig. 6). Fifteen strawberry leaf samples were collected in various commercial production fields. Eleven of the samples were positive for SVBV, the others were negative. Color changes were noted after addition of SYBR green I, with www.nature.com/scientificreports/  www.nature.com/scientificreports/ positive samples turned green and negative samples remained orange. Those observations were consistent with the gel electrophoresis results. These results proved that the PCR and LAMP methods shared high degree of consistency (Fig. 7). In conclusion, the LAMP method developed and optimized in this study is highly specific and much more sensitive than the traditional PCR method, which is completely suitable for field detection of SVBV.

Discussion
At present, there are more than 20 viral diseases that associated with plant decline and yield loss infecting strawberries worldwide 14,22 . Of which the most widespread, prevalent and serious viruses are four aphid-transmitted viruses, including SVBV, SMoV, SMYEV and SCV. SVBV is widely distributed in strawberry growing areas, occurring in China, Japan, America, Brazil, Australia, the Czech Republic, Italy and many other countries 10,23,24 . We have been engaged in strawberry virus detection and disease investigation for more than ten years in China 20,25,26 . An interesting finding is that the SVBV have higher prevalence and dispersion compared to other aphid-transmitted viruses of strawberry and there is a tendency of gradual aggravation in recent years (unpublished data). Cultivated strawberry plants are often symptomless when infected with SVBV alone, while co-infection with other strawberry viruses has the potential to cause serious symptoms in strawberry plants 27 . The primers we used for the PCR assay were designed according to the highly conserved regions (coat protein gene) in the SVBV genomes, which was first used for the American and European sources of SVBV in 1996. After more than 20 years of use these primers are still suitable for most isolates all over the world. We have designed and compared different pairs of primers and found that the SV5508F/SV6606R worked best (data not shown). The brightness of all specific bands are basically consistent with no weak bands appearing, which was in high accordance with other reports 28 . The whole genome of 16 SVBV isolates were sequenced and multiple sequence alignments were performed in this study (Supplementary materials S3). There might be some other highly conserved regions in the SVBV genomes having potential to develop PCR primers that would capture the diversity of all SVBV sequences in the database. Our results will provide the basis for different studies to design primers for different purposes. www.nature.com/scientificreports/ The LAMP assays have been reported for the detection of numerous plant pathogens recently. This method is more reliable, rapid, simple, economical and sensitive than standard PCR on the whole. In the present study, a LAMP assay for the detection of SVBV was developed and found to be more sensitive than conventional PCR technique, as was reported earlier for other pathogens 19,[29][30][31][32][33][34][35] . This method has also been verified to be practical for screening large numbers of field samples, having relevance for field surveying in terms of decisions to keep /manage/ destroy plants.
Strawberry production mainly relies on stolons for vegetative propagation, which provides opportunities for the accumulation and spread of the virus. Once a strawberry plant carries the virus, it will last through the whole growth period. Worse still is that there are no effective measures to prevent strawberry virus disease completely for the moment. Therefore, our recommendation is to eliminate virus-bearing plants as soon as they are found in the field survey if conditions permit. There was evidence that LAMP assay could work with a crude extract 36 . For example, in plant virus diagnosis, it could be possible to use direct crude plant extracts in order to avoid total RNA or DNA extraction, shortening the processing time, allowing the simultaneous analysis of multiple samples, and drastically reducing the total cost for single analysis 37 . Crude extracts were not used in this research, but more attempts will be made in future studies.
When it comes to the cost, labor efficiency, etc., LAMP has great advantages over conventional PCR 25 . Firstly, the amplification reaction can be achieved with a cheap water bath or heater and the results can be interpreted visually without any specific instruments. Secondly, the reagents used in LAMP are also cheaper or equivalent to the standard PCR, including the extraction and testing processes 25 . Furthermore, the LAMP takes less time than PCR method, which is more appropriate in terms of 'time cost' . It has the characteristics of easier operation and the LAMP assay is more labor-saving.  Cloning of SVBV genome sequence. Sixteen isolates in total of SVBV were chosen for sequencing.

Materials and methods
These isolates were obtained from a subset of the 259 strawberry samples described above. All of the samples were obtained from individual plants. Overlapping primers (Table 1) used for PCR amplification were designed based on the published SVBV genome sequences (accession numbers KP311681.1, KR080547.1, HE681085.1 and X97304.1). The extraction of DNA was described above and the PCR reactions were performed in a 25 μL volume with reaction mixtures containing 2.5 μL of 10 × PCR buffer, 1μL of DNA, 2 mM of each dNTP, 0.5 mM of each primer, one unit of LATaq DNA polymerase (TaKaRa, Dalian, China) and brought to volume with ddH 2 O. The amplified genome segments were subsequently cloned. The target fragments were purified according to the instructions of agarose gel purification kit (Aidlab Biotechnologies Co., Ltd). Then the PCR products were cloned into the pBM23 cloning vector using the BMMach1-T1 competent cells and sequenced at Biomed Gene Technology Co., LTD. The kind of sequencing was Sanger's method-Dideoxynucleotide chain termination and the primary instrument was 3730 XL DNA Analyzer. The obtained sequences were aligned with those available in GenBank using the BLAST algorithm (http:// ncbi. nlm. nih. gov/ BLAST/). The complete genome sequences were assembled and analyzed with DNAMAN 7.0 (LynnonBiosoft, Quebec, Canada) and DNASTAR 6.0 (DNASTAR Inc., Madison, WI, USA). Default parameters were used and there was no modification to software default.

SVBV sequence analyses.
All the available genome sequences of SVBV strains in the GenBank database were downloaded and aligned with Clustal X program. Phylogenetic tree based on the genomic nucleotide sequence was performed by neighbor-joining (NJ) method using MEGA7 38 with the best model tested in this www.nature.com/scientificreports/ software and the confidence was estimated by 1000 bootstrap replicates. Recombination was detected with various recombination detection methods implemented in the software RDP5 39 including programs RDP, GENE-CONV, BOOTSCAN, MAXCHI, CHIMAERA, SISCAN and 3SEQ, performed with the default configuration, except that options of circular sequence was selected. Only recombination events detected by at least five different methods were accepted.

Development and optimization of LAMP for detection of SVBV.
According to the sequences of SVBV (AY605663, AY955374, FM867860, JN542480, NC001725) released by NCBI GenBank, the highly conserved region of coat protein gene analyzed by DNAMAN 7.0 was chosen as the target sequence. The primers of LAMP were designed by online software Primer3 Input (http:// bioin fo. ut. ee/ prime r3-0. 4.0/ prime r3/), including four specific primers covering six regions of the CP gene ( Table 2). The LAMP assay was slightly modified according to the method described previously 25,26 . The basic reaction system consisted of 1.6 μM each of the primers SVBV-FIP and SVBV-BIP, 0.  TAC TCG TGA TTC TCA GGT AGA TTG G-3ʹ)  Results of PCR and LAMP assay were analyzed by gel electrophoresis with 1% agarose in Tris acetate-EDTA buffer (TAE: 0.04 M Tris acetate, 1 mM EDTA) and visualized on a UV transilluminator. Additionally, the LAMP outcome could be observed through naked eyes by adding 1 μl SYBR green I nucleic acid dye (Beijing Solarbio Science & Technology Co., Ltd.) to the starting reaction volume of 25 µl, of which the color changed indicating a positive reaction.
Specificity, sensitivity and field applicability of the LAMP assay. The specificity of the LAMP assay was tested using the DNA obtained from SVBV and cDNA from three other important strawberry virus diseases: strawberry mottle virus (SMoV), strawberry mild yellow edge virus (SMYEV) and strawberry crinkle virus (SCV). All of the cDNA controls of the SMoV, SMYEV and SCV had been confirmed accurately to be positive by both RT-PCR and RT-LAMP methods 25,26,41 . Selecting the DNA from healthy plants as the negative control. The positive PCR/LAMP controls were the strawberry leaves infected with SVBV, which had been detected and confirmed before 25,26,41 . To compare the relative sensitivity of LAMP and PCR methods, tenfold serial dilutions (different diluents from 10 0 to 10 -6 ) of SVBV genomic DNA and negative control were prepared as the template of amplification. According to the established detection system, 15 strawberry samples selected randomly from the 259 field samples were tested to evaluate the stability and practicality of this method for field application.  -FIP CAG TGT GAA GTG ATT CCA ACA ATG ATC TTA TCC TTA CTC TCG CAAAG   2  SVBV-BIP CAA ACA AGC TTC TTC AAC AGG ACG AAT TTG TCA GAG TTG TCA   3  SVBV-F 3  CAG AGA AGG CTC TTA CAA ATGA   4  SVBV-B 3  CGA GTT CCC TGT GTA AGA TAG TTA G