DOI: https://doi.org/10.21203/rs.3.rs-1503061/v1
Two Cucumber mosaic virus (CMV) isolates were detected by reverse transcription-polymerase chain reaction from cucumber (Cucumis sativus L.) and cowpea (Vigna unguiculata L. Walp.) plants in Iraq. Analysis of nucleotide sequences of the capsid protein gene place these isolates in the subgroup IB, sharing the highest nucleotide identity (99%) with isolates reported from Iran. This is the first report of CMV subgroup IB in Iraq.
Cucumber mosaic virus (CMV) is the type member of the genus Cucumovirus, family Bromoviridae (Roossinck et al. 1999). It infects over 1200 plant species, most of agriculture importance, causing serious losses worldwide (Zitter and Murphy 2009). CMV comprises three positive-sense (+) RNA segments, namely RNA1, RNA2, and RNA3, which are encapsidated in separate isometric particles (Palukaitis et al. 1992). Proteins encoded by RNA1 and RNA2 are mostly required for the replication of the viral genome, symptoms development, and gene silencing suppression (Ding et al. 1994). RNA3-encoded proteins are essential for viral intercellular movement and particle assembly (Palukaitis et al. 1992). Based on sequence homology, geographical distributions, and serological characteristics, CMV isolates are classified into two subgroups, I and II (Roossinck et al. 1999; Verma et al. 2006). Isolates of subgroup I show a high degree of sequence heterogeneity, sharing 88-99% nucleotide identity, leading to further division two clusters, IA and IB. IA has a worldwide distribution, whereas subgroup IB is more prevalent in East Asia (Roossinck 2002; Palukaitis and García-Arenal 2003). In Iraq, this virus was reported to impact many vegetables, including cucumber and cowpea grown both in glass houses and open fields (CABI 2022). Earlier studies used serological assays and indicator plants to identify CMV in Iraqi crops (e.g. Adhab and Al-Ani 2013). However, the identified CMV isolates have yet to be characterized at the molecular level. The current study analyzes partial nucleotide sequences of two Iraqi CMV isolates, and clarifies their subgroup affiliations.
Symptomatic leaf samples of cucumber and cowpea plants were collected from several fields in north eastern Baghdad Province (Fig. 1a, b). Total RNA was extracted from leaf tissues using RNeasy Plant Mini Kit (Qiagen, Germany) and complementary DNA (cDNA) was synthesized using SuperScript IV reverse transcriptase (Thermo Fisher Scientific, Lithuania). PCR was performed using GoTaq G2 Green Master Mix (Promega, Madison, WI, USA) with primer pairs CPTALLF (5’-YASYTTTDRGGTTCAATTCC-3’) and CPTALLR (5’-GACTGACCATTTTAGCCG-3’) specific to cucumovirus capsid protein (CP) encoding genes (Choi et al. 1999). PCR amplicons (Supplementary Fig. 1) were directly sequenced in both directions (Macrogen, South Korea), and the sequences obtained were deposited in GenBank (IRQ831: MW477481, IRQ832: MZ267789).
Pairwise nucleotide (nt) comparison of the sequences obtained using BLASTn (https://blast.ncbi.nlm.nih.gov/Blast.cgi) showed them to be 100% identical to each other, and have the highest identity (99%) to an isolate of subgroup IB (KT279571) from Iran (Hasanvand and Shams-bakhsh 2017; Supplementary Table 1). Phylogenetic analyses using MEGA X (Kumar et al. 2018) based on aligning the predicted CP amino acid sequence clustered the Iraqi isolates with known members of subgroup IB (Fig. 2). In particular, the Iraqi isolates appear to have common origin with an isolate (KT279571) reported from Iran, providing evidence for CMV spread across borders between neighboring countries.
CMV can threat crops production and affect other Iraqi flora. In this study, the analysis of partial sequences of two CMV isolates confirmed its presence in local varieties of cucumber and cowpea that constitute main cash crops for Iraqi farmers. To our knowledge, this is the first report of CMV subgroup IB isolates in Iraq. The results presented here may support efforts to improve quarantine measures, disease management, and tracking the CMV spread in Iraq agriculture.
The authors declare no competing interests.