Development of viral and viroid detection in Chrysanthemum using real-time RT-PCR and RT-LAMP techiques

Abstract

Chrysanthemum plants (Chrysanthemum x morifolium) are susceptible to virus and viroid infections. To identify causal viruses and viroids that induced symptoms on chrysanthemum, chrysanthemum leaves exhibiting virus- and viroid-like symptoms were surveyed and collected from cultivation areas of Royal Project Foundation in Northern Thailand during 2019 - 2021 and subjected for detection of viruses and viroids. Nine RNA viruses that were reported to infect chrysanthemum, including chrysanthemum virus B (CVB), cucumber mosaic virus (CMV), chrysanthemum stem necrosis virus (CSNV), impatiens necrotic spot virus (INSV), tobacco mosaic virus (TMV), tomato aspermy virus (TAV), tomato spotted wilt virus (TSWV), turnip mosaic virus (TuMV), zucchini yellow mosaic virus (ZYMV) and two viroid including chrysanthemum chlorotic mottle viroid (CChMVd) and chrysanthemum stunt viroid (CSVd) were detected by using reverse transcription polymerase chain reaction (RT-PCR). Moreover, triple-antibody sandwich enzyme-linked immunosorbent assay (TAS-ELISA) was used to detect the DNA virus, tomato yellow leaf curl New Delhi virus (ToLCNDV), using the monoclonal antibody.

Results of virus and viroid detection from total of 242 samples revealed that 3 viruses were detected consisting of CVB, TMV and TuMV and 2 viroids consisting of CChMVd and CSVd were detected by using RT-PCR. Nucleotide sequences of detected viruses and viroids were analyzed and confirmed that they were actual pathogens compared to other isolates and variants in GenBank database. Among all detected pathogens, CVB was the highest incidence.

Furthermore, the incidence of virus and viroid diseases in chrysanthemum of Thailand was lower than 10% considering on detection results. This study is the first report of CVB, TMV, TuMV and CChMVd in chrysanthemum of Thailand. Multiplex nested RT-PCR was developed for detection of CVB, CChMVd and CSVd simultaneously. Among of 16 randomly selected samples, one sample showed coinfections of CVB and CChMVd, and two samples showed multiple infections of all viral pathogens. Therefore, multiplex nested RT-PCR with high sensitivity and specificity can be routinely used for detection and diagnosis of virus and viroid diseases in chrysanthemum. To our knowledge, this is the first detection of mixed infections between virus and viroids in the chrysanthemum of Thailand. Predicted secondary structures of CChMVd and CSVd revealed the distribution of sequence variations. Variations of CChMVd were found on most of the secondary structure whereas sequence variations of CSVd were frequently found in the pathogenic, variation and terminal right domains. Biological indexing was found that CVB, TMV and TuMV induced symptoms on chrysanthemum and other different indicator plants. The slightly flexuous rod-shaped particles approximately 650 – 680 nm long of CVB, rigid rod particles approximately 200 – 300 nm long of TMV and filamentous particles with approximately 750 nm long of TuMV were observed under the transmission electron microscope (TEM). Colorimetric RT loop-mediated isothermal amplification (LAMP) techniques were developed for detecting CVB, TMV, TuMV, CChMVd and CSVd. LAMP primer sets were newly designed and tested for optimization of LAMP detection. All LAMP reactions were achieved at same isothermal incubation at 65°C but the time of incubation was different depended on viruses and viroids consisting of for 30 min (TMV), 45 min (CVB, TuMV and CSVd) and 60 min (CChMVd). Limits of the detection (LOD) was up to 1 fg/ µl for detecting CVB and CSVd and for detecting TMV, TuMV and CChMVd, LOP was up to 10 fg/µl. All 5 colorimetric RT-LAMP and LAMP were not cross reacted to other viruses and viroids. Evaluation of colorimetric RT-LAMP in detecting from actual samples was conducted and found that colorimetric RT-LAMP was effective. Real-time PCR (qPCR) techniques were developed to detect CChMVd and CSVd in chrysanthemum plantlets obtained from meristem tip culture to verify and ensure the production of viroids-free chrysanthemum plantlets. The specific primers were newly designed and the recombinant plasmids of CChMVd and CSVd were used as templates. The standard curves were analyzed and generated. The melting temperature of CChMVd and CSVd were 83.00 and 82.00°C, respectively, whereas the melting temperature of non-specific amplicons from CChMVd and CSVd primers were 67.50 and 73.50°C, respectively. The lowest copies numbers which could be detected for CChMVd was 1x10 copies/µl and CSVd was 1x 10 copies/µl. The efficiency of RT-qPCR was evaluated to detect CChMVd and CSVd from chrysanthemum plantlets. The results showed that all of 18 chrysanthemum plantlet samples were free from CChMVd and CSVd infection.