Application of Multiplex TaqMan Real-Time PCR Assay in Survey of Five Lily Viruses Infecting Lilium spp.

Abstract

Lily symptomless virus (LSV), Lily mottle virus (LMoV), Cucumber mosaic virus (CMV), Shallot yellow stripe virus (SYSV), and Plantago asiatica mosaic virus (PlAMV) are five of the economically important viruses infecting lilies (Lilium spp.) worldwide. In order to prevent the occurrence and spread of these viruses, it is necessary to develop a rapid, effective, and sensitive detection method for the simultaneous detection and specific quantification of these viruses. In this study, specific primers and probes for multiplex TaqMan real-time PCR assays designed from conserved regions of the coat protein sequence of each virus were used for the simultaneous detection of these viruses in lilies (Lilium spp.). The optimal concentration of primers and probes and reaction annealing temperature were 20 µM and 55.9 °C, respectively. The detection limits of the assay were 1.33 × 10², 1.27 × 10¹, 1.28 × 10¹, 2.33 × 10², and 2.01 × 10² copies·μL⁻¹ for LSV, LMoV, CMV, SYSV, and PlAMV, respectively. Specificity was determined using seven viral pathogens of lilies. Variability tests of intra- and inter-assays showed high reproducibility with coefficients of variation <2%. The multiplex TaqMan real-time PCR assay was used to detect these viruses from lily samples in China. In brief, our developed assay showed high specificity, sensitivity, and reproducibility for the simultaneous detection and differentiation of five lily-infecting viruses and can be used for certification and quarantine programs.

1. Introduction

Lilies (Lilium spp.) are among the most important ornamental plants worldwide and are also widely grown for their nutritional and medicinal value in Eastern Asia [1,2,3]. Lily cultivars are usually propagated vegetatively, resulting in virus transmission from generation to generation. Among viruses infecting lilies, Lily symptomless virus (LSV; genus Carlavirus, family Flexiviridae), Cucumber mosaic virus (CMV; genus Cucumovirus, family Bromoviridae), Lily mottle virus (LMoV; genus Potyvirus, family Potyviridae), Shallot yellow stripe virus (SYSV; genus Potyvirus, family Potyviridae), and Plantago asiatica mosaic virus (PlAMV; genus Potexvirus, family Alphaflexiviridae) are five economically important viruses [4,5,6,7,8,9]. LSV-infected plants show mild, pale vein clearing and mottle on leaves, which generally turn yellow fairly quickly [6,7]. LMoV can cause flower-color breaking, leaf mottle, leaf mosaic, chlorotic and yellow streaking, vein clearing, leaf curling, and narrowing [6,7]. CMV-infected lily leaves show chlorotic or yellow spotting, inter-veinal striping or vein-clearing and sometimes malformations [6,7]. Leaves infected with SYSV show yellow stripe symptoms [8]. When infected with PlAMV, leaves of infected plants are often brittle, flower production is declined, and the plants gradually die [9]. Mixed viral infections in lily plants can cause much more severe symptoms, including dwarfism [7]. In order to prevent the occurrence and spread of these viruses, it is essential to establish a rapid, effective, and sensitive detection method for the simultaneous detection and specific quantification of these viruses.

Many diagnostic approaches have been developed to detect viruses, such as serological methods relying on specific antibodies (enzyme-linked immunosorbent assay (ELISA), double-antibody sandwich-ELISA (DAS-ELISA), immunochromatographic strip test, etc.) and molecular biology methods relying on specific primers/probes (reverse transcription-polymerase chain reaction (RT-PCR), real-time PCR, loop-mediated isothermal amplification (LAMP), etc.) [10,11,12,13,14,15,16,17]. ELISA, uniplex RT-PCR, and LAMP have been used to detect lily viruses [18,19,20,21,22]; however, these techniques can only be used to detect one virus per reaction and are time-consuming, laborious, and expensive when several viruses have to be detected in numerous samples [23]. These limitations can be overcome by using multiplex TaqMan real-time PCR assay, which enables the simultaneous detection and identification of several viruses in a single reaction [14]. Multiplex TaqMan real-time PCR has been applied widely for pathogen detection in humans [14,24,25,26,27] and animals [10,28,29,30,31,32,33,34,35]. Recently, this method has also been used to detect viruses in sweet potatoes, pome fruit trees, stone fruit trees, and fig trees [36,37,38,39,40]. However, this method has not been used for the simultaneous detection of LSV, LMoV, CMV, SYSV, and PlAMV in lily plants.

In this study, a set of specific primers and probes were designed for the detection of five economically important viruses (LSV, LMoV, CMV, SYSV, and PlAMV) infecting lily plants. Then, an efficient multiplex TaqMan real-time PCR was developed for the simultaneous detection and differentiation of these lily viruses.

2. Materials and Methods

2.1. Materials

Fresh leaves from lily samples grown in greenhouses at the Chinese Academy of Agricultural Sciences (Beijing, China), which were confirmed to be infected with LSV, LMoV, CMV, SYSV, and PlAMV by RT-PCR and sequencing, were used to establish and optimize the multiplex TaqMan real-time PCR assay.

2.2. RNA Extraction and Reverse Transcription

Total RNA was extracted from lily leaves using an RNAprep pure Plant Kit (Tiangen, Beijing, China), according to the manufacturer’s protocol. First-strand cDNA was synthesized using a SuperScript III reverse transcription kit (Invitrogen, Carlsbad, CA, USA), following the manufacturer’s instructions. Briefly, 5 μg total RNA was combined with 1 µL oligo (dT)₂₀ (50 µM) and 1 µL dNTP mix (10 mM), and nuclease-free water was added to a total volume of 13 μL. The mixture was heated at 65 °C for 5 min and incubated on ice for 1 min. The volume was increased to 20 μL by the addition of 4 µL 5 × first-strand buffer, 1 µL RNaseOUT (40 U), and 1 µL Superscript III reverse transcriptase (200 U). After incubation at 50 °C for 60 min, 1 µL RNase H (2 U) was added, and incubation was continued at 37 °C for 20 min.

2.3. Design of Primers and TaqMan Probes

Sequences of the complete genomes of LSV (accession numbers: HM222522, AJ516059, AM263208, AM422452, and GU440579), LMoV (accession numbers: AB570195, GU440578, AM048875, GU440579, NC_005288, MT795719, and HM222521), CMV (accession numbers: AB506797, AB506800, AJ495841, and AB049568), SYSV (accession numbers: MK127536 and AJ865076), and PlAMV (accession numbers: AB360790, AB360791, AB360792, LC592411, KU159093, LN794199, KT717325, KU159089, and KX245539) were retrieved from the GenBank database, and conserved regions of each virus were identified using ClustalW (Available online: http://clustalw.ddbj.nig.ac.jp/ (accessed on 16 January 2021)). Virus-specific primers and probes were designed according to the identified conserved regions using Beacon Designer 8.0 (Premier Biosoft, Palo Alto, CA, USA) (Table 1).

2.4. Optimization of the Multiplex TaqMan Real-Time PCR Assay

The primers and probes used in these assays were listed in Table 1. All reactions were performed on a Bio-Rad CFX96™ Real-time System (Bio-Rad, Hercules, CA, USA). For uniplex real-time PCR amplifying LSV, LMoV, CMV, SYSV, and PlAMV, the reaction mixture contained 0.4 µL forward primer (20 µM), 0.4 µL reverse primer (20 µM), 0.4 µL TaqMan probe (20 µM), 10 μL 2 × PerfectStart^® II Probe qPCR SuperMix (TransGene, Beijing, China), 2 μL template, and nuclease-free water for a total volume of 20.0 μL. The thermal cycling procedure was initiated at 94 °C for 30 s, followed by 40 cycles at 94 °C for 5 s and 56 °C for 30 s.

Based on the established uniplex real-time PCR, the multiplex real-time PCR was optimized by varying a single parameter while other parameters were maintained. The multiplex real-time PCR was optimized by varying the annealing temperature and the concentration of primers and probes. Different annealing temperatures were set from 52.0 to 62.0 °C (52.0 °C, 52.7 °C, 54.0 °C, 55.9 °C, 58.4 °C, 60.3 °C, 61.4 °C, and 62.0 °C), and the concentrations of primers and probes ranged from 10 to 30 μM (10 μM, 15 μM, 20 μM, 25 μM, and 30 μM). The optimal PCR conditions were selected based on amplification efficiency determined by the Cq (Cycle of quantification) value and the fluorescence intensity. All reactions were conducted in triplicates.

2.5. Construction of Standard Plasmids and Standard Curves

Conventional RT-PCR assays were used to produced the standard fragments of the target viruses using the primers listed in Table 1 (LSV-F/LSV-R for LSV, LMoV-F/LMoV-R for LMoV, CMV-F/CMV-R for CMV, SYSV-F/SYSV-R for SYSV, and PlAMV-F/PlAMV-R for PlAMV). PCR amplification reactions were performed in a final volume of 50 μL containing 1.5 μL each primer (10 μM), 2 μL cDNA, 25 μL 2 × KAPA HiFi HotStart Ready Mix (KAPA Biosystems, Wilmington, WA, USA), and 20 μL double-distilled water. The thermal cycling conditions were as follows: 95 °C for 3 min, followed by 35 cycles of 98 °C for 20 s, 56 °C for 15 s, 72 °C for 30 s, and a final extension at 72 °C for 2 min. The PCR products were electrophoresed in a 2% agarose gel in 0.5 × TBE buffer. PCR products were cloned into the pTOPO vector (Transgen, Beijing, China) to construct recombinant plasmids and sequenced with vector primers at Sangon Biotech (Shanghai, China). The concentrations of the recombinant plasmids were quantified using a Nano Drop (Thermo Scientific, Waltham, MA, USA), and the copy number was calculated as follows: number of copies = [amount (ng) × 6.022 × 10²³]/[length (plasmid + insert) × 1 × 10⁹ × 650] [41]. Standard curves were obtained using serial ten-fold dilutions of the recombinant plasmids of corresponding viruses.

2.6. Specificity of the Multiplex Real-Time PCR

In order to evaluate the specificity of this multiplex real-time PCR assay developed in this study, cDNAs of two other lily viruses, including Strawberry latent ring spot virus (SLRSV; genus Stralarivirus, family Secoviridae) and Arabis mosaic virus (ArMV; genus Nepovirus, family Secoviridae), were used as templates for amplification with this multiplex system. The cDNAs of LSV, LMoV, CMV, SYSV, and PlAMV served as positive controls, and the nuclease-free water was used as a negative template control.

2.7. Analytical Sensitivity

To evaluate the detection limits of the multiplex real-time PCR compared with the uniplex real-time PCRs, 10-fold serial dilutions of the mixture of five recombinant plasmids, ranging from 1.33 × 10⁹ to 1.33 × 10⁰ copies·μL⁻¹, 1.27 × 10⁹ to 1.27 × 10⁰ copies·μL⁻¹, 1.28 × 10⁹ to 1.28 × 10⁰ copies·μL⁻¹, 2.33 × 10⁹ to 2.33 × 10⁰ copies·μL⁻¹, and 2.01 × 10⁹ to 2.01 × 10⁰ copies·μL⁻¹ for LSV, LMoV, CMV, SYSV, and PlAMV, respectively, were used as templates. Samples with a Cq value greater than 35 were considered negatives.

2.8. Reproducibility

To evaluate the reproducibility of the multiplex real-time PCR, three different concentrations of 10⁷, 10⁵, and 10³ copies·μL⁻¹ of standard plasmids were chosen to perform in triplicate on different days. The final coefficient of variation (CV) was calculated to assess intra- and inter-assay variation.

2.9. Survey of Lily Viruses by the Multiplex Real-Time PCR Assay

To further evaluate the efficiency of the multiplex real-time PCR assay, 92 lily samples that showed symptoms of leaf mottle, leaf mosaic, leaf curling, chlorotic and yellow streaking, or flower color breaking were collected from lily production fields in China for the detection of LSV, LMoV, CMV, SYSV, and PlAMV using the developed multiplex TaqMan real-time PCR assay. RNA extraction and reverse transcription were performed using the method described in Section 2.2. Multiplex real-time PCR amplification reactions were performed in a final volume of 20 µL containing 0.4 μL of each of 20 μM LSV, LMoV, CMV, SYSV, and PlAMV primers and probes, 10 µL 2 × PerfectStart^® II Probe qPCR SuperMix (TransGene, Beijing, China), 2 μL template, and nuclease-free water was added to a total volume of 20.0 μL. The PCR conditions were determined as follows: 94 °C for 30 s, followed by 40 cycles at 94 °C for 5 s, 55.9 °C for 30 s. At the same time, the uniplex real-time PCR was performed using the same samples, and the results of the two methods were compared.

2.10. Statistical Analysis

Data analysis and graphing were performed using GraphPad Prism (version 8 for Windows, GraphPad Software, San Diego, CA, USA) and using Jvenn (Available online: http://jvenn.toulouse.inra.fr/app/example.html (accessed on 27 September 2021)) [42].

3. Results

3.1. Optimization of the Multiplex TaqMan Real-Time PCR Assay

Figure 1 shows the optimum concentrations of primers and probes for the multiplex TaqMan real-time PCR assay, which are 20, 30, 20, 20, and 20 μM for LSV, LMoV, CMV, SYSV, and PlAMV, respectively. The results showed the optimum annealing temperatures for LSV, LMoV, CMV, SYSV, and PlAMV amplification were 58.4, 55.9, 58.4, 55.9, and 55.9 °C, respectively (Figure 2). Based on the comparison of reaction performance, 20 μM and 55.9 °C were selected to be the optimal primers and probes concentration and reaction annealing temperature.

3.2. Standard Curves of the Multiplex TaqMan Real-Time PCR Assay

Fragments of the expected size for LSV, LMoV, CMV, SYSV, and PlAMV were amplified specifically using virus-specific primer pairs (Figure 3) and were cloned into pTOPO vector (Transgen, Beijing, China) to obtain recombinant plasmids pTOPO-LSV, pTOPO-LMoV, pTOPO-CMV, pTOPO-SYSV, and pTOPO-PlAMV. The stock of these plasmids had concentrations of 1.33 × 10¹⁰ (pTOPO-LSV), 1.27 × 10¹⁰ (pTOPO-LMoV), 1.28 × 10¹⁰ (pTOPO-CMV), 2.33 × 10¹⁰ (pTOPO-SYSV), and 2.01 × 10¹⁰ copies·μL⁻¹ (pTOPO-PlAMV). Standard curves were constructed using 10-fold serial dilutions of the recombinant plasmids. The results showed that the correlation coefficients (R²) of the five standard curves were above 0.98 (Figure 4), indicating that there was a strong linear relationship between the log₁₀ of the input number of copies and the Cq value of each standard.

3.3. Specificity of the Multiplex TaqMan Real-Time PCR Assay

The results showed that only the positive samples of LSV, LMoV, CMV, SYSV, and PlAMV showed the amplification curve (Figure 5A–E,H), while neither nuclease-free water nor the two non-targeted viruses showed the amplification curve (Figure 5F,G,I).

3.4. Sensitivity of the Multiplex TaqMan Real-Time PCR Assay

Sensitivity testing showed that detection limits were 1.33 × 10² for LSV, 1.27 × 10¹ for LMoV, 1.28 × 10¹ for CMV, 2.33 × 10² for SYSV, and 2.01 × 10² for PlAMV in both multiplex TaqMan real-time PCR and uniplex TaqMan real-time PCR (Figure 6 and Figure 7). These results indicated that the sensitivity of the multiplex TaqMan real-time PCR was the same as that of each uniplex TaqMan real-time PCR assay and that the multiplex TaqMan real-time PCR was suitable for the simultaneous detection of the five viruses.

3.5. Repeatability of the Multiplex TaqMan Real-Time PCR Assay

Different standard plasmids concentrations (10⁷, 10⁵, and 10³ copies·μL⁻¹) were chosen to evaluate the intra- and inter-assay repeatability. The CV values were almost less than 1%, with a few values ranging from 1% to 2% (Table 2) in the intra- and inter-assays, indicating that the assay has high repeatability and reliability.

3.6. Application of Multiplex TaqMan Real-Time PCR Assay in Survey of Lily Viruses

The results of the multiplex TaqMan real-time PCR assay showed that all samples were infected with at least three viruses (Table S1). LSV, LMoV, CMV, SYSV, and PlAMV were detected in 92 (100.00%), 61 (66.30%), 92 (100.00%), 76 (82.61%), and 72 (78.26%) of the 92 samples, respectively (Figure 8A). There were 8 samples co-infected with three viruses, 51 samples co-infected with four viruses, and 33 samples co-infected with five viruses (Figure 8B). All collected samples were further tested using the uniplex TaqMan real-time PCR, and the results were consistent with those of the multiplex TaqMan real-time PCR assay (data not shown). Thus, the multiplex TaqMan real-time PCR assay developed in this study was confirmed to be a rapid and accurate detection method.

4. Discussion

Primer design is the most important step in establishing an efficient multiplex TaqMan real-time PCR assay. Ideally, primers should be specific, have low or no interference in the amplification reaction [38]. In this study, considering the wide genetic diversity of LSV, LMoV, CMV, SYSV, and PlAMV, virus-specific primers were designed according to conserved regions in each viral genome. Using the final primers, five expected amplicons (184 bp for LSV, 88 bp for LMoV, 105 bp for CMV, 80 bp for SYSV, and 153 bp for PlAMV) were produced (Figure 3). And, using this multiplex TaqMan real-time PCR, only the positive samples of LSV, LMoV, CMV, SYSV, and PlAMV showed the amplification curve (Figure 5), revealing that this newly developed assay was highly specific.

The optimization of PCR conditions is essential for efficient PCR amplification. In this study, serial PCRs were conducted to determine the optimal concentration of primers and probes and annealing temperature. The results showed the Cq value and the fluorescence intensity were highest when the concentration of primers and probes and the reaction annealing temperature were 20 µM and 55.9 °C, respectively (Figure 1 and Figure 2). Generally, the presence of more than one pair of primers in the same reaction mix may limit the sensitivity or cause preferential amplification of specific targets [43], and the sensitivity of the multiplex PCR is usually about 10-fold lower than that of a uniplex PCR [36]. Fortunately, the sensitivity of the optimized multiplex TaqMan real-time PCR assay was the same as that of each uniplex assay in our previous study, indicating that a desired primer design and the proper optimization of the multiplex TaqMan real-time PCR assay were developed.

Viral diseases frequently occur in lily plants, and they affect bulb and cut-flower production [4,5,6,7,8,9]. In this study, 92 lily samples in China were tested using the developed multiplex TaqMan real-time PCR assay. The results showed all samples were infected with at least three viruses (Table S1, Figure 8). The high occurrence of viruses infecting lilies in China could be due to the use of virus-infected bulbs for propagation and the lack of preventive virus vector control measures. Hence, it is urgent to use virus-free propagation materials and to take extreme precautions to produce virus-free lily bulbs.

In conclusion, an effective multiplex TaqMan real-time PCR assay was established for the simultaneous detection and differentiation of LSV, LMoV, CMV, SYSV, and PlAMV in lilies. This assay will be useful for routine molecular diagnosis and epidemiological studies of these viruses.

Author Contributions

Validation, L.X. and M.S.; formal analysis, M.S.; investigation, L.X. and M.S.; resources, L.X.; data curation, L.X. and J.M.; writing—original draft preparation, L.X.; writing—review and editing, J.M.; project administration, J.M.; funding acquisition, L.X. and J.M. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the National Key Research and Development Program of China (2019YFD1001805, 2018YFD1000405, and 2019YFD1001002), the National Natural Science Foundation of China (31801899 and 31672196), and the Science and Technology Innovation Program of the Chinese Academy of Agricultural Sciences. This research was conducted at the Key Laboratory of Biology and Genetic Improvement of Horticultural Crops, Ministry of Agriculture, China.

Conflicts of Interest

The authors declare no conflict of interest.

Publisher’s Note: MDPI stays neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Figures and Tables

Figure 1. Optimization of the concentration of primers and probes for multiplex TaqMan real-time PCR assay to detect LSV (A), LMoV (B), CMV (C), SYSV (D), and PlAMV (E).

Figure 2. Optimization of the annealing temperature for multiplex TaqMan real-time PCR assay to detect LSV (A), LMoV (B), CMV (C), SYSV (D), and PlAMV (E).

Figure 3. Determination of specificity of five primer pairs by uniplex RT-PCR to detect LSV (A), LMoV (B), CMV (C), SYSV (D), and PlAMV (E). Lane M, 100 bp plus DNA ladde.

Figure 4. Standard curves obtained by multiplex TaqMan real-time PCR assay for LSV (A), LMoV (B), CMV (C), SYSV (D), and PlAMV (E).

Figure 5. Specificity of multiplex TaqMan real-time PCR. The specific fluorescent signals were detected in the positive samples of LSV (A), LMoV (B), CMV (C), SYSV (D), and PlAMV (E) and mixed samples (H). No cross-reactions were detected from ArMV (F) and SLRSV (G) and ddH20 (I).

Figure 6. Sensitivity of the multiplex TaqMan real-time PCR assay for LSV (A), LMoV (B), CMV (C), SYSV (D), and PlAMV (E). 1–10, ten-fold serial dilutions of plasmid standard, with the concentrations from 1.33 × 109 to 1.33 × 100 copies·μL−1, 1.27 × 109 to 1.27 × 100 copies·μL−1, 1.28 × 109 to 1.28 × 100 copies·μL−1, 2.33 × 109 to 2.33 × 100 copies·μL−1, and 2.01 × 109 to 2.01 × 100 copies·μL−1 for LSV, LMoV, CMV, SYSV, and PlAMV, respectively.

Figure 7. Sensitivity of the uniplex TaqMan real-time PCR assay for LSV (A), LMoV (B), CMV (C), SYSV (D), and PlAMV (E). 1–10, ten-fold serial dilutions of plasmid standard, with the concentrations from 1.33 × 109 to 1.33 × 100 copies·μL−1, 1.27 × 109 to 1.27 × 100 copies·μL−1, 1.28 × 109 to 1.28 × 100 copies·μL−1, 2.33 × 109 to 2.33 × 100 copies·μL−1, and 2.01 × 109 to 2.01 × 100 copies·μL−1 for LSV, LMoV, CMV, SYSV, and PlAMV, respectively.

Figure 8. Detection of five viruses in lily plants in China using multiplex TaqMan real-time PCR assay. (A) Venn diagram illustrating the number of samples infected by multiplex TaqMan real-time PCR assay. (B) Number of samples infected with different viruses.

Table 1

Primers and probes used in this study.

Virus	Primer/Probe	Target Gene	Sequence (5′–3′)	Product Length (bp)
LSV	LSV-F	CP	GCGTCGTATCTAACAACA	184
	LSV-R		GCTCCATTCTCAAACTCA
	LSV-Probe		CY5-CAAGGAACGCCGAACTGCTC-BHQ3
LMoV	LMoV-F	CP	CAGTGAAAGACGAGTATG	88
	LMoV-R		GAGGTGCCATTCTCTATG
	LMoV-Probe		FAM-CAGACCATTCATTGCGAGAGCC-BHQ1
CMV	CMV-F	CP	GACAGTCCGTAAAGTTCC	105
	CMV-R		GATGCAGCGTACTGATAA
	CMV-Probe		Texas Red-TATCCGTTGCCGCCATCTCT-BHQ2
SYSV	SYSV-F	CP	GCTTGGATGGTAACATAAG	80
	SYSV-R		CGTGTGATGATCCTTATTC
	SYSV-Probe		CY5.5-AGAACGACATACAGCAGCCGABHQ2
PlAMV	PlAMV-F	CP	CCAACATCAAGTTCGAAC	153
	PlAMV-R		CGAAGAGGTTTAGGGATC
	PlAMV-Probe		HEX-CGTCTCATTGGCAGTTACTTCGTC-BHQ1

Table 2

Intra- and inter-assay reproducibility of multiplex TaqMan real-time PCR.

Plasmid	Concentration(Copies·μL−1)		Intra-Assay Cq Value			Inter-Assay Cq Value
			Mean	SD	CV%	Mean	SD	CV%
LSV	1.33×	107	17.05	0.29	1.68	17.68	0.02	0.11
		105	25.80	0.30	1.15	26.74	0.07	0.27
		103	31.17	0.07	0.23	31.53	0.18	0.58
LMoV	1.27×	107	17.41	0.02	0.12	16.39	0.21	1.30
		105	23.84	0.12	0.49	23.97	0.22	0.93
		103	30.65	0.16	0.52	31.15	0.13	0.42
CMV	1.28×	107	18.40	0.21	1.14	18.56	0.08	0.41
		105	26.20	0.20	0.76	26.23	0.20	0.76
		103	30.89	0.14	0.44	30.64	0.19	0.61
SYSV	2.33×	107	16.02	0.15	0.94	16.08	0.09	0.54
		105	24.30	0.05	0.19	24.48	0.07	0.27
		103	30.59	0.04	0.14	31.21	0.08	0.27
PlAMV	2.01×	107	15.80	0.28	1.77	15.51	0.12	0.77
		105	24.29	0.46	1.89	23.61	0.24	1.03
		103	31.65	0.55	1.74	32.09	0.08	0.25

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy12010047/s1, Table S1: Survey of viral infection in lily plants in China using the multiplex TaqMan real-time PCR assay.

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