Identification and complete genome sequence of iris potyvirus A, which causes dwarfing and foliar chlorosis with mosaic or mottle disease symptoms on lily (Lilium lancifolium Thunb.) in China

Highlights

• •First report of IrPVA-Anhui natural infect lily plants in China.
• •IrPVA-Anhui is a new member of the genus Potyvirus in the family Potyviridae.
• •It can infect the original host lily back-inoculated healthy plants.

Abstract

Lily plants (Lilium lancifolium Thunb.) exhibiting dwarfing and foliar chlorosis with mosaic or mottle disease symptoms were found in Anhui Province, China. We used high-throughput sequencing of small RNA to survey the virus in the lily cultivation region of Anhui Province. Here, we report the identification and complete genome sequence of the viral agent. It contains 9733 nucleotides, excluding the poly(A) tail, and encodes a polyprotein of 3063 amino acids. The complete polyprotein ORF shows 98.92% amino acid sequence identity with that of iris potyvirus A (GenBank MH898493). Phylogenetic analysis of coat protein sequences placed the viral agent close to members of the genus Potyvirus in the family Potyviridae, and it was therefore provisionally named iris potyvirus A isolate Anhui (IrPVA-Anhui). This is the first complete genome sequence of IrPVA-Anhui from lily plant, for which only a partial sequence from Iris domestica has been reported previously. Comparative analysis of this genome sequence with those of closely related potyviruses identified nine cleavage sites and the conserved motifs typical of potyviruses. Subsequent virus identification was performed using serological assays (ELISA and antibody-based lateral flow assays), molecular methods (RT-PCR), and a pathogenicity test. Virus particles with a length of about 700 nm, similar to viruses in the genus Potyvirus, were observed via transmission electron microscope (TEM). We back-inoculated healthy plants of multiple species to investigate the host range of the virus. It infected the original host, Iris domestica, and Nicotiana benthamiana but not Triticum aestivum, Pisum sativum, Chenopodium amaranticolor, or Datura stramonium. This is the first report of natural IrPVA-Anhui infection of lily plants in China, providing a scientific basis for IrPVA-Anhui control in future lily plantings.

Lily (Lilium lancifolium Thunb.) is a perennial bulb-forming herbaceous plant from the Liliaceae family that is widely grown throughout China. At present, research on lily typically focuses on the anti-tumor, anti-inflammatory, and bacteriostatic activities of its bioactive components (Rina et al., 2020). Most ornamental varieties can also serve as medicinal plants, and some can be used as vegetables (Huiling et al., 2011). Extracts of these plants have long been used in traditional Chinese medicine and have recently been gaining attention in the western world (Wu et al., 2022). Several viruses have been reported to infect lily, including iris potyvirus B (Lin et al., 2022), cucumber mosaic virus and lily symptomless virus (Brierley and Smith, 1944a, 1944b), plantago asiatica mosaic virus (Komatsu et al., 2008), and strawberry latent ringspot virus (Cohen et al., 1995). In addition to lily mottle virus (Bellardi and Bertaccini, 2001; Se and Kanematsu, 2002; Wylie et al., 2012), at least three other potyviruses have been reported to affect lilies (turnip mosaic virus, tulip breaking virus, and lily virus A).

On 11 June 2021, five leaf samples were collected from endemic varieties of lily plants grown in Huoshan County, Anhui Province, China (116°33′E, 31°39′N). Symptoms of infection consisted of plant dwarfing and foliar chlorosis with mosaic or mottle disease symptoms (Fig. 1A). The disease occurred in 5%–7% of the lily plants surveyed, as determined by counting the numbers of symptomatic plants in several fields in Huoshan County. Disease occurrence is associated with a 20% decrease in production. Leaf samples were snap frozen in liquid nitrogen, then stored at −80 °C. Total plant RNA was purified from 3 g of leaf tissue using the TRIzol method following the manufacturer's instructions (Invitrogen). RNA was extracted from individual plant samples and resuspended in nuclease-free water. Agarose gel electrophoresis (1.2%) with ethidium bromide staining was used to check RNA integrity of individual samples. The 260/280-nm absorbance ratio was measured using a BioPhotometer Plus (Eppendorf, Hamburg, Germany) to assess RNA purity (acceptable ratio = 1.8–2.0). Equal amounts of each RNA sample were mixed for analysis.

Fig. 1.

Symptoms of diseased lilies in the field (A), lilies inoculated with IrPVA-Anhui (B), leaf samples collected from the same lily-planting areas in 2022 (C), Nicotiana benthamiana inoculated with IrPVA-Anhui (D), and symptomless Iris domestica inoculated with IrPVA-Anhui (E), particles morphology of IrPVA-Anhui using electron microscopy negative staining, bars: 100 nm (F), Samples tested by antibody-based lateral flow assay (G).

A small RNA (sRNA) library was constructed using a TruSeq Small RNA Library Prep Kit (Illumina), then deep sequenced by BGI-ShenZhen (China) on the Illumina HiSeq 2000 platform (Illumina, San Diego, CA, USA). Sequencing produced 17,117,098 clean reads ranging in length from 18 to 28 nucleotides (nt). Velvet was used to assemble the sRNAs, with the minimal overlap length (K-mer) required to join two small RNAs into a contig set to 17 nucleotides (Kreuze et al., 2009; Wu et al., 2010). Assembly of the sequenced sRNAs using Velvet generated 2763 contigs. BLASTn and BLASTx were used to compare the contigs with entries in the non-redundant nucleotide and protein databases at GenBank. BLASTx (e-value cut-off = 10⁻³) identified 13 contigs, ranging in length from 117 to 1200 bp, as being related to partial genomic sequences of iris potyvirus A isolate Won (MH898493). To obtain the full genome of IrPVA-Anhui, multiple primers (Table S1) were designed using the sequences and relative positions of contigs mapped to the genomic RNA. The primers were used to amplify the gaps between various contigs, as well as the terminal untranslated regions. The contigs were then joined together after further Sanger sequencing (TSINGKE, Beijing, China), reverse transcription PCR (RT-PCR), and validation of ambiguous nucleotide sequences from one individual sample. The amplicons were cloned into the pMD18-T vector (Takara, Dalian, China) for automated DNA sequencing, and three clones of each PCR fragment were sequenced. Rapid amplification of cDNA end PCR (RACE-PCR) (Takara Biotechnology, Dalian, China) was used to obtain the 5′ and 3′ ends of the viral genome. ORF Finder was used to predict the open reading frames (ORFs) in the sequence. MEGA 7.0 with the Neighbor Joining algorithm and 1000 bootstrap replicates was used to construct a phylogenetic tree of coat protein amino acid sequences from IrPVA-Anhui and 31 other members of the Potyviridae; the results showed that IrPVA-Anhui clustered together with members of the genus Potyvirus (Fig. 2).

Fig. 2.

Phylogenetic tree constructed by the neighbor-joining method in MEGA 7, showing the relationships among IrPVA-Anhui and 31 other members of the family Potyviridae based on the coat protein amino acid sequence. Numbers at the nodes are bootstrap values from 1000 replicates, and bar lengths represent evolutionary distances.

The complete IrPVA-Anhui genome sequence (9733 nt) was submitted to GenBank under accession number MZ604653. The IrPVA-Anhui genome nucleotide sequence is 97.54% identical to and shares 99% coverage with that of IrPVA isolate Won. Bioinformatics analysis predicted that the IrPVA-Anhui genome encodes one large ORF of 3063 amino acids with a molecular weight of 345.8 kDa. The 5′ untranslated region (UTR) of the virus is composed of 119 nt preceding the initiation codon, and the 3′ UTR contains 323 nt downstream of the polyprotein, preceding the poly(A) tail. Comparison with 22 polyproteins from members of the genus Potyvirus showed that the deduced IrPVA-Anhui polyprotein has high amino acid sequence identity with those of other potyviruses. It has the highest identity (98.92%) to IrPVA isolate Won, followed by 58.04% identity to lily virus Y (Table 1). The sequence identity of the IrPVA-Anhui polyprotein to that of lily virus Y is significantly below the 82% threshold used to discriminate between species, indicating that IrPVA-Anhui is an indeed a new species of the genus Potyvirus. The conserved RdRp, CP, and HC-Pro-domains of the polyprotein share the highest amino acid sequence identities (99.51%, 99.14% and 98.39%) with those of IrPVA isolate Won (Table 1). Like other potyviruses, it is predicted to be cleaved into 10 mature proteins at nine proteolytic cleavage sites (Adams et al., 2005) (Fig. 3). The mature proteins of IrPVA-Anhui are as follows: P1 (305 aa), HC-Pro (456 aa), P3 (349 aa), 6K1 (53 aa), CI (635 aa), 6K2 (53 aa), NIa-VPg (188 aa), NIa-Pro (243 aa), NIb (515 aa), and CP (266 aa). The highly conserved motif 3068GAAAAAA3075 and a small ORF (PIPO) encoding 76 aa were found to be embedded in the P3 protein (Chung et al., 2008). Some motifs characteristic of members of the genus Potyvirus include an HX₈DX₂₈GXSG (aa215–256) motifs exist in P1(Valli et al., 2007). An HXCX₂₇CX₂C (aa 327–360) motif, a PTK motif (aa 612–614, aphid-transmission-associated motif), and an KITC motif (aa 354–357) in HC-Pro (Atreya and Pirone, 1993). An EPYX₇SPX₂L (aa 793–807) motif in P3(Riechmann et al., 1992), GXXGXGKS (aa 1247–1254), VLLLEPTKPL (aa 1267–1276), DECH (aa 1336–1339), IKMSATPP (aa 1362–1369), LVYV (aa 1414–1417), VATNIIENGVTL (aa 1465–1476), and IQRLGRVGR (aa 1512–1520) motifs in CI; an HX₃TX₂GHCG (aa 2180–2190) motif in NIa- Pro (Dougherty et al., 1989); the conserved residues SLKAEL (aa 2450–2455), ADGSQFD (aa 2527–2533) and GDD (aa 2630–2632, RNA-dependent polymerase activity) in NIb; and a DAG (aa 2805–2807, aphid transmission) motif in CP (Chen et al., 2020; Li et al., 2018).

Table 1.

Genome length and amino acid sequence identities of iris potyvirus A isolate Anhui with equivalent regions of other members of the genus Potyvirus.

Virus name	GenBank accession	Genome length (nt)	Sequence identity (%)
			Polyprotein	RdRp	CP	HC-Pro
Iris potyvirus A	MH898493	9707	98.92	99.51	99.14	98.39
Lily virus Y	MF543013.1	9811	58.04	69.93	75.11	62.67
Thunberg fritillary mosaic virus	AJ885005.1	9723	55.96	64.29	68.24	56.32
Paris virus 1	MN549985.1	10,066	56.37	66.50	72.10	57.47
Platycodon mild mottle virus	NC_055503.1	9556	53.66	67.57	66.09	51.49
Lily mottle virus	AJ564636.1	9644	52.87	64.53	69.70	52.53
Plum pox virus	HF585098.1	9784	52.15	67.08	70.39	51.03
Clover yellow vein virus	KU922565.1	9585	51.28	67.98	65.24	49.54
Bean yellow mosaic virus	D83749.1	9532	51.35	68.47	67.81	49.43
Asparagus virus 1	KJ830760.1	9741	49.18	65.85	69.53	48.85
lupinus mosaic virus	eu847625.2	10,113	49.52	63.64	67.81	49.20
Chilli veinal mottle virus	aj237843.3	9711	46.92	61.27	73.82	47.13
Carrot thin leaf virus	JX156434.1	9491	49.04	66.01	71.24	46.67
Japanese yam mosaic virus	AB027007.1	9760	49.83	66.75	69.96	49.20
Turnip mosaic virus	AF169561.2	9835	49.77	66.75	66.09	49.43
Scallion mosaic virus	AJ316084.1	9324	50.49	66.59	70.39	51.49
Narcissus yellow stripe virus	AM158908.1	9650	49.90	66.83	71.24	49.66
Tobacco etch virus	M11458.1	9494	50.00	62.65	68.24	49.08
Potato virus A	AJ296311.1	9585	50.00	60.90	66.52	47.13
Sunflower chlorotic mottle virus	GU181199.1	9965	48.41	66.83	66.09	47.13
Iris severe mosaic virus	KT692938.1	10,423	45.39	63.97	59.66	39.64
Potato virus Y	X97895	9701	47.55	64.48	67.38	46.21

Fig. 3.

Genome organization of IrPVA-Anhui. The cleavage sites (`/') for generation of 10 mature proteins were predicted by comparison with closely related potyviruses. The position of each gene is indicated, showing nucleotide coordinates.

To determine the causal agent(s) of the disease, symptomatic leaves (n = 4) were collected for electron microscopy negative staining. Virus particles with a length of about 700 nm, similar to viruses in the genus Potyvirus, were observed via transmission electron microscope (TEM), suggesting the presence a potyvirus(es) (Fig. 1F).

Samples of lily viral disease were collected by amplification of partial genome sequences using reverse transcription PCR (RT-PCR). cDNA was synthesized using an Oligo (dT)23 primer or random hexamer primers (TaKaRa Biotechnology Dalian Co., Ltd). The RT reaction contained 0.1 mg of total RNA and Moloney murine leukemia virus (M-MLV) reverse transcriptase (TaKaRa) according to the manufacturer's instructions. Samples were identified using RT-PCR to obtain amplicons of 244 bp using primers IrPVA-1F/IrPVA-1R and 479 bp using primers IrPVA-2F/IrPVA-2R (nt 3710–4188 and nt 6301–6544 in IrPVA-Anhui) (Fig. 4A). Sap extracts from leaf samples were tested using an antibody-based lateral flow assay and direct antigen-coated ELISA with polyclonal antibodies specific to potato virus Y, tobacco mosaic virus, and cucumber mosaic virus (Agdia Inc., USA) and a monoclonal antibody for the potyvirus group. The samples were positive for the potyvirus group and potato virus Y (Fig. 1G). It is well known that some ‘virus-specific’ potyvirus antibodies yield cross-reactions with other potyviruses .

Fig. 4.

RT-PCR products generated using the IrPVA-1F/1R and IrPVA-2F/2R primers. PCR products (1 µL of a 25 µL reaction) were resolved by electrophoresis on a 1.0% agarose gel and visualized by ethidium bromide staining. A: lane M is the molecular weight marker (DL2000), lane 1 shows the 244-bp amplicon obtained using primers IrPVA-1F/1R, and lane 2 shows the 479-bp amplicon obtained using primers IrPVA-2F/2R. PCR results from lily, Iris domestica, Nicotiana benthamiana, Triticum aestivum, Pisum sativum, Chenopodium amaranticolor, and Datura stramonium inoculated with IrPVA-Anhui, as well as lily samples collected in 2022. B: lane M is the molecular weight marker (DL2000), lanes 1 and 8 are lily inoculated with IrPVA-Anhui, lanes 2 and 9 are T. aestivum inoculated with IrPVA-Anhui, lanes 3 and 10 are P. sativum inoculated with IrPVA-Anhui, lanes 4 and 11 are lilies collected in 2022, lanes 5 and 12 are N. benthamiana inoculated with IrPVA-Anhui, lanes 6 and 13 are C. amaranticolor inoculated with IrPVA-Anhui, lanes 7 and 14 are D. stramonium inoculated with IrPVA-Anhui, and lanes 15 and 16 are I. domestica inoculated with IrPVA-Anhui. Lanes 1–7 and 15 show the 244-bp amplicons, and lanes 8–14 and 16 show the 479-bp amplicons.

A pathogenicity test on healthy lily plants grown under greenhouse conditions was performed to complete Koch's postulates. Sap extracts of leaf samples were used to mechanically inoculate lily plants at the rosette stage. After 15 days, inoculated lilies showed symptoms similar to those of the original infected plants (Fig. 1B). Emerged systemic leaves were then tested by RT-PCR, confirming the presence of IrPVA-Anhui and satisfying Koch's postulates (Fig. 4B, lane 1) (Robinson, 1992).

A host range experiment was then performed by inoculating the virus onto lower leaves of Iris domestica, Nicotiana benthamiana, Triticum aestivum, Pisum sativum, Chenopodium amaranticolor, and Datura stramonium. Fifteen days post inoculation, only Iris domestica (Figs. 1E and 4, lanes 15 and 16) and Nicotiana benthamiana (Figs. 1D and 4B, lanes 5 and 12) were infected by IrPVA-Anhui, based on RT-PCR and Sanger sequencing results. The original host Iris domestica remained symptomless after inoculation (Fig. 1E). No positive amplicons were obtained from T. aestivum, P. sativum, C. amaranticolor, or D. stramonium post inoculation (Fig. 4B, lanes 2, 3, 6, 7, 9, 10, 13, 14). Neither systemic infection by the virus nor a hypersensitive response was observed on inoculated leaves of these species, showing that they are not hosts of IrPVA-Anhui. On 11 June 2022, symptomatic samples collected in the field also tested positive for IrPVA-Anhui (Figs. 1C and 4B, lanes 4 and 11).

To our knowledge, it is the first report of IrPVA-Anhui associated with dwarfing, foliar chlorosis, and mosaic or mottle disease symptoms on lily. This plant virus causes a 20% decrease in lily production and significant economic losses. Lily is a direct host of the virus, and IrPVA-Anhui control is therefore important for protecting lily production. Selection of uninfected plants in the propagation stock or production of healthy plant material through meristem tip culture and tissue culture propagation aided by thermotherapy and/or chemotherapy are the most effective means of control.

CRediT authorship contribution statement

Fang Wang: Funding acquisition, Methodology, Software, Investigation, Formal analysis, Writing – original draft. Dankan Yan: Data curation, Writing – original draft. Kelei Han: Visualization, Investigation. Zhengliang Gao: Resources, Supervision. Chao Ma: Software, Validation. Ying Chen: Visualization, Writing – review & editing. Xianxun Bao: Conceptualization, Resources, Supervision, Writing – review & editing. Cheng Li: Funding acquisition, Writing – review & editing.

Declaration of Competing Interest

The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Fang Wang reports financial support was provided by Natural Science Foundation of Anhui Province (2008085MC74). Fang Wang reports financial support was provided by Open Fund of the State Key Laboratory of Tea Plant Biology and Utilization (SKLTOF20210116). Dankan Yan reports financial support was provided by National Natural Science Foundation of China (32001872). Cheng Li reports financial support was provided by Key Research and Development Plan of Anhui Province (202204c06020011). 1. There has added a support in acknowledgement as bellow: Acknowledgement This study was supported by the Natural Science Foundation of Anhui Province (2008085MC74), the Open Fund of the State Key Laboratory of Tea Plant Biology and Utilization (SKLTOF20210116), National Natural Science Foundation of China (32001872), Key Research and Development Plan of Anhui Province (202204c06020011). 2. In order to operat the systerm to submitted paper, Fang Wang has been the corresponding author to carry out the system operation before, but in fact, Cheng Li and Xianxun Bao are the corresponding authors of this article as manuscrript submitted.

Data availability

• No data was used for the research described in the article.