Development of methodologies for virus detection in soybean and wheat seeds

Graphical abstract


Methods detail
The protocol described for RNA extraction of immature seeds of jatropha (Jatropha curcas), mustard (Brassica sp.) and rice (Oryza sativa) seeds is a simple, quick and efficient protocol for RNA extraction and downstream applications [1]. To use it for virus detection in soybean (Glycine max) and wheat (Triticum aestivum) seeds, some improvements are needed to be done in the maceration step. Two viruses of quarantine importance, Soybean mosaic virus (SMV), family Potyviridae, genus Potyvirus [2] and Wheat streak mosaic virus (WSMV), family Potyviridae, genus Tritimovirus [3,4] were chosen to check if the protocol is suitable for application in their identification in seeds. The molecular tools: RT-PCR test for WSMV detection [3] and the RT-PCR for SMV detection [5] adapted to RT-qPCR were compared to other techniques and selected to perform virus detection with the RNA obtained from seeds.

Sample preparation
Soybean plants were mechanically inoculated with an isolate of SMV (named 165.09 GenBank: KC331990) intercepted at Plant Quarantine Laboratory of Embrapa Genetic Resources and Biotechnology. Wheat plants of cv. Guabiju were also mechanically inoculated with WSMV (isolate 915 GenBank: KC152463.1 from Passo Fundo, Brazil). Plants were cultivated in greenhouses to obtain seeds. Seeds were harvested and stored at 4 8C until the maceration procedure began. In addition, seeds of soybean (several accessions), wheat cv. BRS Guabiju and BRS Guamirim, triticale cv. BRS Saturno and BRS Ulisses and maize hybrid HS201 from healthy plants were collected for RNA extraction procedure. Prior to maceration, the wheat and triticale seeds were incubated with water in order to soften the tissues.

Maceration
(1) Put an amount of 0.5 g of wheat/triticale seeds into a mortar and subsequently cover the seeds with about 20 mL of distilled water. Cover the mortar with a paper towel and wait at least 12 h at room temperature to start the maceration in order to allow the seeds to absorb water. (2) Pulverize 1.2 g of dry soybean seeds, 0.5 g of dry maize seeds and the previously incubated wheat and triticale seeds in liquid nitrogen. Put the seed powder into 50 mL pre-chilled polypropylene (Falcon) tube. RT-PCR is used for detection of Wheat streak mosaic virus from wheat seeds and RT-qPCR for detection of Soybean mosaic virus from soybean seeds. The method may be tested for other viruses, however, pre-validation will be needed.  confirmed by agarose gel electrophoresis for WSMV, amplification profile and melting curve analysis of qPCR for SMV. The PCR products of the expected size (469 bp for SMV and 948 bp for WSMV) can be sequenced to confirm the identity of the virus. The quality of RNA obtained from healthy and infected seed samples, their concentrations and results of virus detection are summarized in Table 1 and Fig. 1.

Validation
In order to use the method for routine procedures, we validated the method according to EPPO [6] guidelines. Because only one sample of wheat seeds was positive to WSMV, thirteen recombinant plasmids with the coat protein fragment of WSMV were diluted up to 10 À3 in wheat/triticale seed RNA to simulate the conditions of the test (presence of potential inhibitors for PCR test in the seeds). Two independent experiments with conventional PCR were performed as described above. Five isolates of SMV had the RNA extracted from leaves using the RNeasy mini kit (Qiagen) and the cDNA prepared as described above. The cDNAs were mixed with soybean seed RNA (due to the presence of potential inhibitors for PCR test in the seeds) and diluted up to 10 À2 for subsequent qPCR. Two independent experiments were performed for duplicate samples with the same conditions previously described. Results are available in Table 2 and Fig. 2. In addition, the seed samples with positive results for WSMV and SMV were submitted to the traditional quarantine scheme of planting and Elisa test from leaves and similar results were obtained ( Table 2).

Background information
About 230 plant virus and viroid that cause diseases on plants are seed-transmitted [7]. There are economically important virus species, such as SMV and WSMV, which are transmitted through soybean and wheat seeds respectively. Traditionally, the scheme for detection of SMV and WSMV includes seed planting, in order to observe typical symptoms and the Elisa tests from leaf samples or reverse transcription PCR (RT-PCR) with specific primers [4,5,8]. This scheme is time-consuming (3-4 weeks to observe symptoms) and might not have enough sensibility to detect the viruses in low [ ( F i g . _ 2 ) T D $ F I G ]  concentrations. Currently, there are some reports of detection of RNA viruses through commercial kits in tomato and pepper seeds [9,10]. However, individual seeds were used, which may not be always suitable for quarantine or seed certification analyses that deal with large amount of seeds. In addition, soybean seeds are rich in oil and wheat seeds are rich in starch. Our previous analysis using different commercial kits for these seeds showed low quantity and quality of RNA.
The results showed that the concentration (in ng) of all samples was within the acceptable range with good quality (low concentration of proteins), varying among samples. This variation could had occurred due to the nature of samples (storage conditions, age of seeds, among others) and also occurs when commercial kits are used for RNA extraction from more suitable tissues, such as leaves. The virus detection was performed and results were the same as obtained by planting-Elisa method. No falsepositive result was obtained and the false-negative results were in the acceptable range [6]. Unfortunately, non-specific bands, different in size compared to positive control, were visualized in the PCR for WSMV (Fig. 1A, lanes 7 and 8). These bands were sequenced and had the highest identity with plant genes. However, it was not possible to improve the specificity of the method without an increase in the level of false-negative results. On the other hand, the bands that were slightly different in size in the qPCR for SMV ( Fig. 2A) may reflect the genetic differences in the virus population. The presence of recombination events and mutations in the genome of the SMV are considered responsible for the emergence of virulent strains [11]. In addition, defective virus frequently occurs in certain proportion in a viral population [12].
The total time for analysis, including cDNA synthesis and qPCR, might be less than seven hours (nine hours for conventional PCR). This case study reveals the potential to be an alternative to the current scheme of planting and testing from symptomatic tissues. A single sample from a batch of seed could be tested against certain viruses, reducing the time and the space required for analysis. This is especially important for analysis in places where it is impossible to plant the seeds, or when the time for detection is short, like for inspection in harbors, airports or border areas. Sometimes seeds present reduced viability, especially when infected by viruses or other pathogens, and the planting is useless for analysis.
The seeds infected by SMV and WSMV collected for RNA extraction did not show symptoms such as mottling or reduced size. Due to the potential presence of other virus species transmitted by seeds in soybean, like Cowpea mild mottle virus (genus Carlavirus), Tobacco ringspot virus (genus Nepovirus), Tobacco streak virus (genus Ilarvirus) and in wheat, like Barley stripe mosaic virus (genus Hordeivirus) among others [7], the extraction of RNA from seeds may be very useful for seed certification and quarantine. To improve this approach, other specific tests against a broad range of seed-transmitted viruses have to be developed and/or validated in order to keep the same reliability. In these experiments, the amount of 1.2 g corresponded to approximately 8-10 soybean seeds. The amount of 0.5 g corresponded to approximately 20-25 entire wheat seeds or 40-50 longitudinally cut half seeds.