Liu et al. Phytopathology Research (2021) 3:6 https://doi.org/10.1186/s42483-021-00082-1 Phytopathology Research RESEARCH Open Access A new potyvirus isolated from Pennisetum alopecuroides with the potential to infect cereal crops Xuedong Liu1,2†, Xi Chen1†, Sijia Liu1, Kaitong Du1, Pei Wang1, Tong Jiang1, Mengji Cao3, Xiangdong Li4, Zaifeng Fan1 and Tao Zhou1* Abstract Pennisetum plants (Pennisetum alopecuroides L.), displaying a dwarfing phenotype along with delayed flowering and mosaic symptom on leaves, were found in Beijing, China. Flexuous filamentous particles with a size of approximate 15 × 850 nm were observed in symptomatic leaves via transmission electron microscopy. Deep sequencing of small RNAs (sRNA) from symptomatic leaves and analysis of sRNA populations were then conducted to determine the genome sequence of the viral agent in diseased plant tissues. It showed that the viral agent had one positive-sense and single-stranded RNA genome, which consisted of 9717 nucleotides (nts) excluding poly(A) tail. The complete viral genome contained a large open reading frame, encoding a polyprotein of 3131 amino acids (aa). Sequence comparison and phylogenetic analysis demonstrated that the viral agent belonged to the genus Potyvirus in the family Potyviridae. In the cladogram it was most closely related to johnsongrass mosaic virus, sharing 72% nt and 65% aa sequence identity. This viral agent was provisionally named pennisetum alopecuroides mosaic virus (PalMV). Subsequently, it was confirmed that PalMV is the causal agent of this new disease in P. alopecuroides by Koch’s postulates and reverse transcription-polymerase chain reaction analysis. Moreover, maize, millet, wheat, sorghum and rice plants were experimentally infected by PalMV via rub inoculation. Consequently, we proposed that PalMV could be a potentially dangerous virus threating a wide range of cereal crops. Keywords: Pennisetum alopecuroides, Potyvirus, PalMV, Cereal crops, Koch’s postulates, Maize, Millet Background yield, extensive adaptability and low cost, P. alopecur- Pennisetum grass (Pennisetum alopecuroides L.), a re- oides has been considered as an alternative and promis- newable prolific herbaceous plant species in the family ing plant species that can adapt to different ecological Gramineae, can serve as a source for feed, knitting, environment (Kang et al. 2018). medicine, paper-making and biofuel production (Wang The Potyviridae is the largest family of plant-infecting et al. 2017). The annual dry matter yield of Pennisetum RNA viruses, including a number of agriculturally and grass ranges from 40 to 50.2 t/ha, which is significantly economically important viral pathogens, and resulting in higher than those of sugarcane and corn (13–21 t/ha) serious disease epidemics in a wide range of cultivated (Tang et al. 2019). Due to its rapid growth, high biomass plants (Revers and Garcia 2015; Wylie et al. 2017). In terms of genomic organization, members of the family * Correspondence: [email protected] Potyviridae consist of monopartite and bipartite plant vi- †Xuedong Liu and Xi Chen contributed equally to this work. ruses with single-stranded and positive-sense RNA ge- 1 State Key Laboratory for Agro-Biotechnology, and Ministry of Agriculture nomes, ranging in size from 8.2 kb to 11.3 kb with an and Rural Affairs, Key Laboratory for Pest Monitoring and Green Management, Department of Plant Pathology, China Agricultural University, average size of 9.7 kb. The flexuous filamentous particles Beijing 100193, China with no envelope are 680–900 nm long and 11–20 nm in Full list of author information is available at the end of the article © The Author(s). 2021 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. Liu et al. Phytopathology Research (2021) 3:6 Page 2 of 10 diameter encompassed by a single core capsid protein 2019). Moreover, genomic information obtained from (Wylie et al. 2017). NGS data would accelerate the development of routine Currently, the family Potyviridae comprises 12 defini- diagnostics and expedite the progress of epidemiological tive genera (i.e., Arepavirus, Bevemovirus, Brambyvirus, surveys aimed at carrying out disease control or eradica- Bymovirus, Celavirus, Ipomovirus, Macluravirus, Poace- tion strategies (Adams et al. 2009; Hadidi et al. 2016). virus, Potyvirus, Roymovirus, Rymovirus and Tritimo- In this study, we report a new viral disease on P. alo- virus) distinguished by the host range, genomic features pecuroides. The causing agent of this disease was provi- and phylogeny (Wylie et al. 2017; Walker et al. 2020). sionally named as pennisetum alopecuroides mosaic The species classification criteria, based upon their open virus (PalMV), which was verified by Koch’s postulates. reading frames (ORF) or polyprotein products, are nor- NGS analysis of sRNA reads and 5′ and 3′ RACE PCR mally accepted as < 76% nucleotide (nt) identity and < enabled assembly of the complete genomic sequence of 82% amino acid (aa) identity. If the complete ORF se- PalMV. Sequence comparison and phylogenetic analysis quence is not available, analogical criteria can be applied indicated that PalMV was most closely related to JGMV. to the coat protein (CP) coding regions and their prod- Moreover, maize, millet, wheat, sorghum and rice were ucts (Wylie et al. 2017; Lefkowitz et al. 2018). experimentally infected by PalMV via rub inoculation. The Potyvirus is the type genus of the family Potyviri- PalMV was proposed as a member of the genus Poty- dae. Most potyviruses infect dicots, and only a few can virus on the basis of the biological and physiochemical infect monocots (Revers and Garcia 2015; Wylie et al. characteristics determined by technologies available. 2017; Lefkowitz et al. 2018). The following potyviruses have been identified infecting monocots, including maize Results dwarf mosaic virus (MDMV), sorghum mosaic virus A filamentous virus was observed in diseased P. (SrMV), zea mosaic virus (ZeMV), sugarcane mosaic alopecuroides tissues by transmission electron microscopy virus (SCMV), johnsongrass mosaic virus (JGMV), (TEM) cocksfoot streak virus (CSV) and pennisetum mosaic In June 2016, P. alopecuroides showing a dwarf pheno- virus (PenMV), which all belong to the SCMV subgroup type along with leaf chlorosis and delayed flowering were (Barnett 1992; Shukla et al. 1992; Seifers et al. 2000; found in Haidian District in Beijing (Fig. 1), and the Gotz and Maiss 2002; Fan et al. 2003). Potyviruses usu- plants were suspected to be infected with potential viral ally induce longitudinal chlorotic or necrotic streaks in agents. To verify this hypothesis, TEM was utilized to the leaves of monocotyledonous plant species, and chlor- visualize virus particles within the diseased plant tissues. otic vein banding, mosaic mottling, necrosis, or distor- Uniquely, flexuous filamentous particles with a size of tion of leaves in dicotyledonous plant species along with approximate 15 × 850 nm were observed via TEM (Add- alterations on flowers, fruits and seeds (Revers and Gar- itional file 1: Figure S1), resembling those of potyviruses cia 2015). (Wylie et al. 2017; Yang et al. 2018; Cui and Wang 2019; In the past few years, the rapid development of next- Lan et al. 2019). generation sequencing (NGS) technology has advanced our ability to investigate diseases of unknown etiology Deep sequencing of sRNA and determination of the full- and expedited the identification and characterization of length genomic sequence of the viral agent new viral pathogens (Adams et al. 2009; Al Rwahnih Total RNA was isolated from symptomatic P. alopecur- et al. 2009; Kashif et al. 2012; Hadidi et al. 2016; Ramos- oides leaf samples and used for high-throughput sequen- Gonzalez et al. 2017; Lan et al. 2019). Compared with cing of sRNAs. Approximately, 18 million reads ranging some traditional techniques, such as enzyme-linked im- from 18 to 25 nts in length were obtained from sRNA li- munosorbent assay (ELISA), PCR assays and brary (Fig. 2a). These reads were mapped to the viral hybridization methods, the NGS technology does not re- genome in sense and antisense orientations, meanwhile quire any virus-specific reagents (i.e., primers, probe or 21 and 22 nts virus-derived small interfering RNAs antibody) of the suspected pathogens. Due to antiviral (vsiRNAs) accumulated to high levels (Fig. 2b). To defense, a mass of 20–22 nts small RNA (sRNA) frag- examine the genomic distribution of vsiRNAs, vsiRNA ments are abundant in virus-infected plant tissues (Wu sequences were mapped along the viral genome. The et al. 2010). The sRNA reads are trimmed and assem- single nucleotide resolution maps indicated that vsiR- bled into larger contigs, with a combination of NGS ana- NAs from both polarities were almost continuously but lysis and PCR-based targeted resequencing to confirm heterogeneously distributed throughout the entire gen- some low coverage areas. NGS data provide a wide range ome, and the region corresponding to the CP gene con- of sequences in search of the pathogens involved in vari- tained more hotspots (Fig. 2c). ous diseases (Al Rwahnih et al. 2009; Kashif et al. 2012; Subsequently, sRNA reads were trimmed and assem- Roy et al. 2015; Ramos-Gonzalez et al. 2017; Lan et al. bled into larger contigs using CLC Genomic Workbench Liu et al. Phytopathology Research (2021) 3:6 Page 3 of 10 Fig.