A Distinct Strain of Chickpea chlorotic dwarf virus Infecting Pepper in Oman

Abstract

During a field survey in 2011, pepper (Capsicum annum) plants showing symptoms suggestive of geminivirus infection were observed in three fields in the Al-Sharqiya region of Oman. Symptoms observed included upward leaf curling leading to cupping and stunting with 15 to 25% disease incidence in surveyed fields. Total DNA was extracted from the leaves of seven symptomatic plants and subjected to rolling circle amplification (RCA). The RCA product was digested with several restriction endonucleases to obtain unit length of ~2.6 to 2.8, typical of geminivirus. Out of seven samples, only four yielded a product of ~2.6 kb in size by KpnI digestion. The fragments were cloned in pUC19 and sequenced. The partial sequences of four isolates were >95% identical to each other at the nucleotide (nt) level and thus only one isolate (P-25) was fully sequenced, determined to be 2,572 nt in length, and its sequence deposited in GenBank (KF111683). The P-25 sequence showed a genome organization typical of a mastrevirus, with four open reading frames (ORFs), two in virion-sense and two in complementary-sense. The virion and complementary-sense ORFs were separated by a long intergenic region, containing a predicted hairpin structure with the nonanucleotide sequence (TAATATTAC) in the loop, and a short intergenic region. An initial comparison to all sequences in the NCBI database using BlastN showed the isolate to have the highest level of sequence identity with isolates of the dicot-infecting mastrevirus Chickpea chlorotic dwarf virus (CpCDV). Subsequent alignments of all available CpCDV isolates using the species demarcation tool (2) showed the isolate P-25 to share between 83.6 and 90.3% identity to isolates of CpCDV available in databases, with the highest (90.3%) to CpCDV strain A originating from Syria (FR687959) (3). Amino acid sequence comparison showed that the predicted proteins encoded by the four ORFs of P-25 (coat protein [CP], movement protein [MP], replication associated protein A [RepA], and RepB) share 91.5, 88.2, 89.1, and 90.8% amino acid sequence identity, respectively, with the homologous proteins of the CpCDV isolate from Syria. Based on the recently revised mastreviruses species and strain demarcation criteria (78 and 94% whole genome nt identity, respectively) proposed by Muhire et al. (2), the results indicate that isolate P-25 represents a newly identified strain (strain F) of CpCDV. The presence of CpCDV in symptomatic pepper plants was further confirmed by Southern blot hybridization technique using digoxygenin (DIG) labeled probe prepared from CpCDV isolate P-25. The genus Mastrevirus consists of geminiviruses with single component genomes that are transmitted by leafhoppers. Mastreviruses have so far only been identified in the Old World and infect either monocotyledonous or dicotyledonous plants (1). To our knowledge, this is the first report of a mastrevirus on the Arabian Peninsula and the first record of pepper as host of CpCDV. Recently, several begomoviruses of diverse geographic origins have been found infecting vegetable crops in Oman. The propensity of geminiviruses to evolve through recombination may lead to evolution of recombinant CpCDV with new host adaptability. Due to extensive agricultural/travel links of Oman with rest of the world, there exists high probability for the spread of this virus. References: (1) M. I. Boulton. Physiol. Mol. Plant Pathol. 60:243, 2002. (2) B. Muhire et al. Arch. Virol. 158:1411, 2013 (3) H. Mumtaz et al. Virus Genes 42:422, 2011.