The First Report of Alfalfa mosaic virus Occurrence on Pepper and Tomato Crops Based on Partial Nucleotide Sequence analysis of the Virus Coat Protein Open Reading Frame from Hormozgan Province

Emerging plant viral diseases account for serious threats to agricultural crop yields and food security in many 
parts of the world. Alfalfa mosaic virus (AMV), a member of the Alfamovirus genus within the Bromoviridae family, 
exhibits a wide host range, infecting more than 150 plant species, including economically important crops such as 
alfalfa (Medicago sativa), lettuce (Lactuca sativa), potato (Solanum tuberosum), tomato (Licopersicon esculentum), 
pepper (Capsicum annuum) and bean (Phaseolus vulagris). AMV was initially documented infecting alfalfa in the 
Fars and Tehran provinces in 1968, subsequently spreading to all alfalfa-growing regions of Iran country (Zainadini 
et al. 2005). Serological assays have traditionally identified AMV strains, with molecular characterizations also 
being conducted on Iranian AMV isolates (Golnaraghi et al. 2004; Massumi & Hosseini Pour 2007; Massumi et al. 
2012; Mangeli et al. 2012; Pourrahim & Farzadfar 2015). However, a number of Iranian AMV isolates have also 
been characterized molecularly (Mangeli et al. 2012; Pourrahim & Farzadfar 2015). Notably, based on ELISA tests 
and molecular examinations, Mangeli and colleagues identified AMV in various weeds, alfalfa, and vegetables such 
as potatoes and peppers across different Iranian regions (Mangeli et al. 2019). A greenhouse survey in Bandar 
Abbas, Iran, in spring 2023 revealed severe leaf chlorotic spots, yellowing symptoms, and fruit necrosis in tomato 
(Haji-Abad) and pepper (Bandar-Abbas) plants (Figure 1). To confirm AMV infections, total RNA was extracted 
from five symptomatic and one asymptomatic sample of tomato and pepper plants using TRIzol reagent (Sinaclone), 
followed by reverse transcription-polymerase chain reaction (RT-PCR) assays with a set of specific AMV primers 
(AMV-R, AMV-F) targeting a 780-nucleotide segment of the coat protein gene (Massumi et al. 2012). PCR 
products of the expected size (approximately 780 bp) were obtained from all of the symptomatic samples (five 
samples of tomato and pepper plants), while no amplified products were generated from any of the healthy leaf 
samples of tomato and pepper plants. Amplified fragments from one sample of each plant were gel purified using a 
PCR purification kit (Qiagen Co.) and sequenced directly using a paired-end sequencing strategy (Sinohe Co. Iran). 
Multiple alignments of the obtained nucleotide sequences (corresponding to a part of CP gene) with 15 AMV 
isolates obtained from GenBank showed the highest identity (97.9%. for IR-To-01 isolate and 98.3% for IR-Pep-03 
isolate) with an Italian AMV strain See-1 isolated from Sechium elude and the lowest identity (95.8% for IR-To-01
and 95.4% for IR-Pep-03) with an Iranian AMV isolate (IRWS2) from Wisteria sinensis. Phylogenetic analysis 
grouped Iranian isolates with European strains, while, the other AMV isolates (including Iranian, Asian and 
American isolates) were grouped into a separate clade (Figure 2), suggesting a possible introduction through seed 
trade. Further PCR assays targeting other viral groups, using four primer pairs, including a degenerate primer pair of 
potyviruses Nib1 / Nib3R (Gibbs and Mackenzie 1997); a degenerate primer pair of begomoviruses primer BC 
(Deng et al. 1994) / primer181V (Rojas et al. 1993) and Tobamovirus degenerate primer pair TobamodF/TobamodR 
(Li et al. 2018), yielded no positive results, suggesting no mixed infection of AMV with other viruses. Given the 
economic importance of tomatoes and related industries in southern Iran and the potential of emerging viruses which 
can rapidly spread and infect tomato crops, leading to reduced fruit production and quality, understanding the 
epidemiology and genetic variations of emerging viral diseases such as AMV is crucial for effective control 
measures. Ongoing research aims to elucidate genetic variations, pathogenicity, host range, and distribution of AMV 
in tomato and pepper plants under greenhouse and open-field conditions in Iran. Based on the results of nucleotide 
sequencing, based on a small part of the virus envelope protein from tomato (Haji Abad) and pepper (Bandar Abbas) 
under different cultivation conditions in Iran, particularly in Hormozgan province, a major center for greenhouse and 
outdoor tomato and pepper cultivation.