Areca palm (Areca catechu L.) is extensively cultivated as a cash crop in South and Southeast Asian countries (Khan et al., 2023). In China, areca palm cultivation is predominantly concentrated in Hainan Province. As of 2018, the total cultivated area of areca palm in Hainan had reached approximately 0.11 million hectares, significantly contributing to the income and wealth of numerous farmers in the province.
Unfortunately, the cultivation of areca palm faces several threats, with one of the most significant being yellow leaf disease (YLD). YLD was first observed in areca palm in India in1914 and is characterized by leaf yellowing, alongside with a range of other manifestations (Yu et al., 2015). In Hainan, an estimated 40% of areca palm acreage is affected by YLD, leading to substantial economic losses (Khan et al., 2023).
The etiology of YLD remains a subject of ongoing debate. While earlier studies implicated phytoplasmas as the causative agent (Nayar & Seliskar, 1978), recent investigations have proposed a closterovirus named areca palm velarivirus 1 (APV1) as a primary candidate (Wang et al., 2020, Zhang et al., 2022, Yu et al., 2015). Furthermore, two potyviruses have been recently identified in areca palm, potentially contributing to a disease closely resembling YLD (Yang et al., 2018, Yang et al., 2019).
Ormycoviruses constitute a newly proposed group of RNA viruses (Forgia et al., 2022). Unlike typical RNA viruses, however, the RNA-dependent RNA polymerase (RdRp) of ormycoviruses does not contain the conventional GDD catalytic triad (te Velthuis, 2014). Instead, they have NDD, GDQ, or less frequently, SDD, HDD or ADD (Forgia et al., 2022).
In this short communication, we report the detection and molecular characterization of a putative ormycovirus in areca palm. Interestingly, this ormycovirus is frequently detected in YLD-affected areca palm but is apparently absent in healthy areca palm.
In our quest to understand the etiology of Yellow Leaf Disease (YLD), we conducted transcriptome sequencing experiments. Areca palm leaf samples displaying characteristic YLD symptoms (Fig. S1) were collected in Baoting County, Hainan Province, China, in July 2022. Total RNA was extracted from a mixed pool of these samples using the CTAB method (Li et al., 2008). Subsequently, a cDNA library was prepared using the ‘Illumina TruSeq Total RNA with rRNA Sample Preparation Kit’ and sent to Novogene (Beijing, China) for paired-end transcriptome sequencing on the Illumina Novaseq™ 6000 (Illumina, San Diego, CA, USA) instrument (150bp × 2). Raw RNA-Seq datasets were processed to eliminate low-quality reads and adapter sequences, accomplished through fastp (Chen et al., 2018). Reads shorter than 36 nt were systematically discarded. De novo assembly of the resulting 45869010 clean reads was conducted using the Trinity assembler 2.8.5 (Haas et al., 2013) and the assembled contigs were subjected to BLASTx searches against the NCBI NT and NR databases.
Upon reviewing the BLASTx results, a contig was identified to has the RdRp of plasmopara viticola lesion associated ormycovirus 7 (PvlaOMV7 (Chiapello et al., 2020) as its top match, exhibiting an amino acid sequence identity of 31.38% and covering 59% of the reference sequence. This contig spans 3,085 nucleotides in length and was represented by 555,480 unique reads, strongly suggesting the presence of a putative ormycovirus in our samples. For simplicity, we refer to this putative ormycovirus as areca palm yellow leaf-associated ormycovirus (APYLaOMV) hereafter.
Most ormycoviruses possess a second genome segment that potentially encodes the capsid protein of the virus in addition to a segment encoding RdRp (Forgia et al., 2022). However, despite extensive analysis of our deep sequencing data, we were unable to identify any contigs corresponding to this second segment of APYLaOMV. Additionally, all our attempts to detect this segment using conventional PCR proved unsuccessful. As a result, we presume that APYLaOMV is monopartite virus in this study, although we acknowledge the possibility that its second segment may have eluded detection.
The 5’ and 3’ sequences of APYLaOMV were then obtained by rapid amplification of cDNA ends (RACE) experiments. Following verification through conventional RT-PCR, cloning, and sequencing experiments, the final sequence was deposited in GenBank under the accession number OR204700.
To explore the molecular characteristics of APYLaOMV, we identified putative open reading frames (ORFs) within the APYLaOMV genome using the ORF Finder program available at the NCBI. Subsequently, we aligned the RdRP of APYLaOMV with those of related ormycoviruses using the MAFFT algorithm (Katoh & Standley, 2013) implemented in PhyloSuite 1.23 (Xiang et al., 2023). Pairwise identity matrixes were generated based on the MAFFT alignments using SDT 1.2 (Muhire et al., 2014) using as input MAFFT alignments. Furthermore, a maximum likelihood (ML) phylogenetic tree was constructed to infer the relationships between APYLaOMV and other ormycoviruses. The tree was reconstructed using IQ-TREE (Nguyen et al., 2014) under the VT + F + G4 evolutionary model, which was determined by ModelFinder (Kalyaanamoorthy et al., 2017). To assess the reliability of the inferred ML tree, we subjected it to ultrafast bootstrapping with 10000 replicates.
The complete sequence of APYLaOMV spans 3063 nucleotides (nts), excluding the 3' poly(A) tail. A single ORF extending from nt 17 to 3,007 (depicted in Fig. 1A) was detected, which encodes a putative RdRp consisting of 997 amino acid residues. As the RdRp of prototypic RNA viruses (te Velthuis, 2014), the RdRp of APYLaOMV has the typical A-B-C motifs (Fig. 1B). However, in line with the observations made from other ormycoviruses, the motif C of the APYLaOMV RdRp is characterized by an NDD triplet instead of the conventional GDD.
In terms of sequence identity, the RdRp of APYLaOMV shares 30.3% sequence identity with the RdRp of PvlaOMV7 and exhibits amino acid identities ranging from 20.3–25% when compared to the RdRps of 11 other ormycoviruses (as depicted in Fig. 1C).
Ormycoviruses have been categorized into three genera, namely alphaormycovirus, betaormycovirus, and gammaormycovirus, based on sequence identity and phylogenetic analyses (Forgia et al., 2022). Our phylogenetic analysis aligns with this classification and suggests that APYLaOMV belongs to the proposed genus alphaormycovirus. However, it's important to note that the genus alphaormycovirus forms two distinct clades in our phylogenetic tree, denoted as Clade 3 and Clade 4 in Fig. 2. This suggests that further refinements may be needed for the classification of alphaormycoviruses.
To investigate the relationship between APYLaOMV and YLD, we conducted RT-PCR to detect APYLaOMV in 43 distinct areca palm samples, comprising 23 displaying typical YLD symptoms and 20 healthy ones. The reverse-transcription primer R1 and the PCR primers F1 and R1 (refer to Table S1) were used in this study. A fragment of the expected size (~ 630 bp) was successfully amplified from 16 of the 23 samples displaying YLD symptoms (data not shown). However, this fragment was not detected in any of the 20 healthy samples. To validate the results of the RT-PCR, three of the PCR amplicons from samples with YLD symptoms were subjected to Sanger sequencing using specific primers. All three amplicons were found to correspond to the targeted region of APYLaOMV.
As the name implies, all known hosts of ormycoviruses to date have been fungi (Chiapello et al., 2020, Forgia et al., 2022). Given the close associations between plants and fungi, it is reasonable to hypothesize that APYLaOMV may be infecting a fungus that colonizes areca palm, despite the absence of any visible signs of fungal infection in our areca palm samples. To investigate this possibility, we conducted screening of total DNA extracts from the areca palm leaf samples, in which APYLaOMV has been detected, using the fungal-specific 18S rRNA PCR primers ( White et al. (1990). As anticipated, we did not detect the presence of any fungal species in these samples. Consequently, APYLaOMV may indeed be a plant virus. This notion is supported by the fact that several groups of viruses originally found exclusively in fungi have later been discovered to also exist in plants. In addition, the abundance of clean reads corresponding to APYLaOMV also argues against the idea that they were derived from an endophytic fungus.
In summary, we have identified a putative ormycovirus named APYLaOMV in areca palm, marking the first documented instance of its presence in this plant species. The fact that not all YLD-affected areca palm samples test positive for APYLaOMV suggests that it may not be the sole causal agent of YLD. However, it does not preclude the possibility that this virus could potentially contribute to the manifestations of YLD under specific conditions. This notion aligns with the observation that APYLaOMV is absent in healthy areca palm samples and the fact that YLD is a complex disease, exhibiting a wide range of symptoms (Khan et al., 2023). Consequently, our findings introduce a new candidate for consideration in the ongoing exploration of the mechanisms underlying YLD in areca palm.