Molecular Detection of Three Viruses Infecting Mallow Plants in Golestan Province

The genus Malva (family Malvaceae) includes species of herbaceous plants which are often pluriannual and very common in borders and boundaries of cultivated fields, making them proper reservoirs for plant pathogens. They are most commonly used as ornamental plants, although they may be used as a food resource and remedy for various diseases. Common mallow (Malva sylvestris) is the most important Malva species which is used as garden flower as well as widely recognized for food and medicinal purposes.  Malva sylvestris are reported to be affected by several viruses from different genera including malva vein clearing virus (MVCV, Potyvirus), alfalfa mosaic virus (AMV, Alfamovirus), malva-associated soymovirus 1 (MaSV1, Soymovirus) cucumber mosaic virus (CMV, Cucumovirus), and cotton leaf curl Gezira virus (CLCuGeV, Begomovirus). In this study, we attempted to identify important viruses infecting mallow plants and compare mallow isolates with other sequences in the GenBank.

In 2022-2023, twenty samples of mallow plants with viral-like symptoms on the leaves were collected from the urban landscape of Golestan province. Total RNA was extracted by DENAzist Total RNA Isolation Kit from fresh, infected mallow leaves. All samples were analyzed by RT-PCR with specific primer pairs for the detection of CMV, AMV, and MVCV. The amplified fragments in expected size of coat protein (CP) gene of each virus were visualized under UV light in agarose gel after electrophoresis, then amplified fragment of one isolate of each virus was bi-directionally sequenced. Obtained sequences were compared to data available in GenBank. The phylogenetic trees of viruses were constructed based on the nucleotide sequences of the coat protein gene using the neighbor-joining method by MEGA11.

All three viruses were detected in some symptomatic samples collected from Golestan province. The most typical symptoms in positive samples were mosaic, vein clearing, and leaf malformation. Of a total of 20 sampled plants, MVCV, CMV and AMV were detected by RT-PCR in nine, five and two samples, respectively. An amplicon of each virus was selected and sequenced in both directions. BLASTn analysis of the sequenced data confirmed that the PCR-amplified fragments belonged to MVCV, AMV, and CMV. Phylogenetic analysis based on the nucleotide sequence of CP gene of MVCV (n=21) including Iranian and GenBank isolates showed that all isolates are divided into six groups: G1 to G6. All Iranian isolates along with the isolate from the Netherlands were placed in group G5. The phylogenetic tree placed the CMV sequences (n=51) into two distinct phylogroups I and II; the obtained isolate CMV-IR-Ma clustered together with isolates from Iran, Netherlands, South Korea, Japan, China, Hungary, Australia, France and the USA into group II. According to the results of this study, AMV isolates (n=17) can be divided into two groups, I and II. Group I which includes isolates from Canada, China, Italy, Spain, Argentina, Australia and the USA, was divided into six subgroups. The obtained isolate AMV-IR-Ma was clustered in subgroup IB with a Chinese isolate (HZ) and forms a common branch with isolates (Ir-VM, Ir-WS, and Ir-WS2) from Iran. This is the first report of mallow (Malva sylvestris) infection with CMV and AMV in Iran using RT-PCR. In addition, mixed infection with two (MVCV+CMV and MVCV+AMV) or all three viruses (MVCV+CMV+AMV) was also confirmed in some samples. The phylogenetic trees showed that most of the viral isolates were not grouped according to their geographic locations. This suggests the dissemination and spread of these viruses through infected seeds. In Iran, M. sylvestris is a common weed found in fields, waste grounds, roadside verges and gardens. Considering the potential non-persistent aphid transmission as well as mechanical transmission, virus-infected mallows can act as a natural reservoir, thereby posing a threat to other ornamental plants and crops. Since the studied viruses were not detected in the other symptomatic plants, the observed symptoms can be caused by physiological disorders such as nutrient deficiencies, the presence of different viral strains, or other unknown and undetected viral species.

This study provides new knowledge on the diversity and molecular characteristics of viruses in mallow plants (M. sylvestris) affected by the viral disease. The information obtained from this study can be helpful in improving management strategies for disease caused by these viruses in Iran. Albeit M. sylvestris is a host of these viruses, but more comprehensive research on other viral species that may infect this plant need to be conducted. Weed management could be an effective way to eliminate inoculum sources of these viruses.