Incidence, Molecular Detection, and Partial Nucleotide Sequencing of Some Viruses Causing Fig Mosaic Disease (FMD) on Fig Plants in Egypt

Fig mosaic disease (FMD) is a viral disease that poses a significant danger to Egypt's fig-producing economy. During the two growing seasons 2017 and 2018, fig leaves and fruits displaying a variety of symptoms linked with fig mosaic disease (FMD) were collected and differentiated from the most famous fig-growing governorates in Egypt, Mersa Matruh, Ismailia, and Giza. Symptomatic samples were tested for the presence of fig mosaic virus (FMV), fig leaf mottle-associated virus 1 (FLMaV-1), fig leaf mottle-associated virus 2 (FLMaV-2), fig mild mottle-associated virus (FMMaV), fig latent virus 1 (FLV-1), fig fleck-associated virus (FFkaV), and fig cryptic virus (FCV) using reverse transcription-polymerase chain reaction (RT-PCR) with specific primers. Three viruses were detected in mixed infections and showed positive results. FMV was detected with infection rate 49% followed by FLMaV-2 with infection rate 21.8% and FLMaV-1 with infection rate 10.9%, respectively, whereas all tested samples were negative for the other viruses. According to the sequence and phylogenetic analysis, the Egyptian FMV isolate was closely related to other FMV isolates, particularly the Argentina ones (Acc. No. KP796424), with 99% identity. While FLMaV-1 showed more than 98% identity with reference isolate FLMaV-1 (Acc. No. LN873219), on the other hand, the isolate of FLMaV-2 showed 100% identity with reference FLMaV-2 isolate (Acc. No. FJ473383) based on phylogenetic analysis. Because fig output in Egypt is expanding, our findings suggest that greater attention should be paid to improving the phytosanitary condition of fig trees in Egypt.


Introduction
, and it is very important in Egypt [3]. Tunisia, respectively, with FMV being the most prevalent [10][11][12]. In Tunisia, FLV-1 was found in all examined regions, in both symptomatic and asymptomatic trees. In Iran, [13] discovered three viruses in fig trees: FLV-1 (dominant), FLMaV-1, and FMV. In Lebanon, Egypt, and western Saudi Arabia, 4-5 viruses were found in fig trees with FLMaV-1 being the most common followed by FMV [11,14,15]. In 2008, FBV-1, the only DNA virus reported in fig to date, was discovered in an FMD tree in the United States [16]. e virus was discovered to be one of the most common in diverse fig-growing regions throughout the world [2,[17][18][19]. e identification of fig mosaic virus (FMV) recently classified as a member of the genus Emaravirus, as the primary agent of FMD in 2009 and marked a watershed moment in the disease's genesis [20,21] [20,[24][25][26]. In recent years, partial or complete nucleotide sequences of additional viruses, most likely from the Partitiviridae (luteovirus-like) and Caulimoviridae (badnavirus-like) families, have been discovered in infected fig plants [16,17,27].
Various molecular approaches are currently highly effective in detecting viruses or viral particles in plants [23,28,29]. RT-PCR was therefore an accurate, rapid, and dependable technology used by several authors to detect FMD viruses [2,10,13,15,16,19,30]. In recent years, the use of nucleotide fragment sequence analysis and comparison with different fig virus isolates from around the world has significantly aided in the detection of fig mosaic disease viruses and understanding the ecology of the disease risk, emergence, and dynamics of fig mosaic disease [10,19]. As a result, the purpose of this study was to isolate and identify the major viruses that have affected some Egyptian local fig varieties due to fig mosaic disease, as well as to determine the virus presence using RT-PCR and to compare the nucleotide sequence analysis of the observed viruses to homologs reported in the GenBank database.  Figure 1). Each leaf sample was assessed based on the presence or absence of FMD symptoms. e proportion of leaves with certain symptoms among those polled was used to assess disease incidence.

Total Nucleic Acid
Extraction. RNA was extracted from leaf vein tissues from symptomless samples as well as leaf samples with mosaic-like symptoms. Using the Plant Total RNA Mini Kit (Real Biotech Corporation, Banqiao, Taiwan) (RBC Labs), total RNA was extracted from 100 mg of plant leaves. e leaves were crushed in liquid nitrogen, macerated in 500 μl of grinding buffer, and then purified with two cycles of filter and RB column followed by ethanol precipitation.
e RNA pellet was dissolved in RNase-free water and kept at −20°C until it was utilized in one-step RT-PCR [19].  Table 1). e PCR mixture (25 μl) contained 12.5 μl of 1-step PCR ready-mix (2X), 1.25 μl of RT enhancer, 0.5 μl (10 μM) of forward and reverse primer for each virus (Table 1), 3 μl of template RNA, and 0.5 μl of verso enzyme mix built up to the final volume with 6.75 μl of nuclease-free water. Amplifications were carried out in a TECHNE thermocycler (TC-512). e RT-PCR programme of FMV, FLMaV-1, FLMaV-2, FMMaV, FCV, and FFkaV consisted of a reverse transcription step at 50°C for 30 minutes, followed by 2 minutes at 94°C and 35 cycles of 94°C denaturation for 30 seconds, 55-58°C annealing for 30 seconds, 72°C primer extension for 35 seconds, and final extension at 72°C for 7 minutes. However, in the case of FLV-1 amplification cycles consisted of a reverse transcription step at 50°C for 30 minutes, followed by 2 minutes at 94°C and 35 cycles of 95°C denaturation for 45 seconds, 52°C annealing for 30 seconds, 72°C primer extension for 1 minute, and final extension at 72°C for 10 minutes.

Agarose Gel Electrophoresis Analysis.
e amplified RT-PCR products were evaluated in 1% agarose gel in 1XTBE buffer at 120 V for 1 hour, stained with ethidium bromide (0.5 μl/ml), and photographed using Gel Doc XR-170-8170 (Bio-Rad, California, USA). e molecular weight of the PCR products was determined by comparing them to the DNA marker ladder weight of 100 bp (Invitrogen, Gothenburg, Sweden).

Gel Extraction, DNA Sequencing, and Sequence Analysis.
e RT-PCR products' fragments were extracted from agarose gel using QIAGEN's QIAquick Gel Extraction Kit according to the manufacturer's instructions. e FMV, FLMaV-2, and FLMaV-1 partial purified genes were bidirectionally sequenced using forward and reverse fig virusspecific primers (Table 1) (Macrogen, Seoul, Korea). Nucleotide sequences were assembled and processed, and various sequences of related viruses infecting figs were acquired from GenBank and compared using BLAST (NCBI database) and DNAMAN software (Lynnon BioSoft. Quebec, Canada). Following multiple sequence alignments, the alignments were used to reconstruct phylogenetic trees for the Egyptian isolates of FMV, FLMaV-1, and FLMaV-2 using maximum-likelihood bootstrap analyses with 1,000 replicates, which were performed to estimate the support for inferred phylogenies.

Disease Symptoms.
In the fields for figs (four-five years), a field survey examination was performed to check for any visual viral disease symptoms of cultivars, Sultany, kommathri, El-Adasy, and some unknown varieties cultivated in three Egyptian governorates, Mersa Matruh, Ismailia, and Giza throughout the growing seasons April and May of 2017 and 2018, revealing that viral disease symptoms were quite extensive and diversified. Almost two-thirds of the fruit trees examined visually had one or more viral symptoms. ese symptoms were observed on leaves and/or fruits, including mosaic, chlorotic ringspot, deformation, chlorosis, mottling, chlorotic blotching, chlorotic blistering, vein clearing,     FMD symptoms were seen on leaves and fruits in all of Egypt's studied cultivars; however, the symptoms were of varying forms and severity. Spain, England, Albania, Cyprus, Greece, Turkey, Yemen, Algeria, Morocco, Mexico, Syria, Lebanon, Australia, South Africa, Italy, Japan, America, Saudi Arabia, Iran, China, and Palestine have all reported the same findings of the diseases existence and spread [11,31,32,34,35]. It was confirmed that global FMD attack frequencies and severity vary by season and are not constant over time. ese variances are mostly determined by the prospecting year's meteorological circumstances, the surveyed varieties, and the tree's vigorous state [36]. e accuracy of RT-PCR in detecting viruses associated with FMD is demonstrated in this study. Our findings revealed the presence of three viruses, FMV, FLMaV-1, and FLMaV-2, among the seven viruses examined that have been associated with FMD. e findings corroborate those of Ref. [15], who found FMV, FLMaV-1, FLMaV-2, and FMMaV in fig leaf samples, and Ref. [30], who found FMV, FLMaV-1, FLMaV-2, FMMaV, FFKaV, FCV-1, and FLV-1 in fig leaf samples during a preliminary survey conducted on a small number in some Egyptian governorates. In comparison with our findings, all 137 tested trees from all cultivars were infected by at least one virus, with FMV and FLMaV-2 being the most prevalent viruses with incidence rates of 49% and (a)  International Journal of Microbiology 5

Discussion
21.8% in all tested plants, respectively, followed by FLMaV-1 with an incidence rate of 10.9% in all tested plants. Our findings corroborate prior reports from a number of Mediterranean countries [1,2,7,23,34,[37][38][39][40][41][42]. e RdRp and HSP70 genes have highly essential sequence and sequence analysis information for the taxonomy of Bunyaviridae and closteroviridae members. Ref. [31] advocated that FMV be classified as genus Emaravirus, FLMaV-1 as genus Closterovirus, and FLMaV-2 as genus Ampelovirus, despite the fact that their findings were contradictory (based on BLAST analysis of sequence from the four RNA segments). Ref. [39] proposed that FMV is a negative-sense single-stranded RNA virus, whereas FLMaV-1 and FLMaV-2 are positive-sense single-stranded RNA viruses from the same family. e partial nucleotide sequence alignment of the FMV-Egyptian isolate showed 99%, 97%, 96%, 95%, and 91% similarities with Argentina, Japan, Canada, Costa Rica, and Greece isolates, respectively, while the partial nucleotide sequence alignment of the FLMaV-1 Egyptian isolate showed 99%, 97%, 92%, 92%, and 88% similarities with Saudi Arabia, Italy, Turkey, Montenegro, and Tunisia isolates, respectively. However, the partial nucleotide sequence alignment of the FLMaV-2 Egyptian isolate showed 100%, 98%, 93%, 92%, and 88% similarities with Italy, Saudi Arabia, Tunisia, Algeria, and Syria isolates, respectively. ese findings are consistent with those of Ref. [43], who stated that sequencing analyses revealed that FMV and FLMaV-1 Spanish isolates shared 89-93% identity with other Mediterranean isolates of the same viruses reported in the GenBank database. On the other hand, Ref. [3] stated that sequence and phylogenetic analysis revealed that the Egyptian FMV isolate was closely related to other FMV

Conclusion
In this paper, we describe the prevalence of viruses that naturally infect

Conflicts of Interest
e authors declare that they have no conflicts of interest.

Supplementary Materials
Multiple