Abstract: Alfalfa mosaic virus is one of the most important viruses infecting potato worldwide. Genetic comparison between Coat Protein (CP) gene of AMV isolate infecting potato in Upper Egypt (AMV-Assiut) and worldwide isolates was carried out in this study. The AMV-Assiut isolate shared similarity in CP gene ranged from 90-95 and 95-97% in nucleotide and amino acid sequences of CP gene, respectively. The AMV-Assiut shared the highest similarity with Egyptian AMV isolates (Wady Elnatron) and AMV isolate from Croatia in case of nucleotide and amino acid, respectively. Phylogenetic analysis showed that AMV isolates tend to cluster into two main groups, with additional clustering of AMV isolates in each group into two subgroups, supporting the hypothesis of existence two main strains of AMV. No clear geographical or host origin structure was found among AMV isolates.
INTRODUCTION
Potatoes are considered as one of the most important vegetable crops in Egypt (El-Helaly et al., 2012). It is being infected by several viruses causing great losses in potato production (Wangai and Lelgut, 2001). More than 25 viruses are reported to infect potato worldwide (Beemstar and Rozendaal, 1972). Among these viruses Alfalfa mosaic virus (AMV) is considered as one of the most important viruses infecting potato worldwide (Jaspars and Bos, 1980). It causes diseases in many economically important crops families including Solanaceae and Fabaceae (Hiruki and Miczynski, 1987).
Alfalfa mosaic virus (AMV) is the type member of genus Alfamovirus in Bromoviridae family of plat viruses (Parrella et al., 2000). The AMV particles are composed of icosahedral capsids measuring 30-57 nm in length and 18 nm in diameter (Thole et al., 1998). The genome of AMV consists of three single strand RNA molecules of plus sense polarity, conventionally numbered RNA 1-3 of decreasing size (Xu and Nie, 2006), encapsidated into B, M and T components, respectively (Jaspars, 1985). It is transmitted by aphids in non-persistent way (Hull, 1969). Sixteen species of aphids including Myzus persicae can transmit AMV (McDonald and Suzuki, 1983; Moreira et al., 2010). The AMV can also be transmitted by potato pollen and true seed (Crill et al., 1971; Zitikaite and Samuitiene, 2008).
Alfalfa mosaic virus (AMV) has a wide host range (Parrella et al., 2000; Al-Saleh and Amer, 2013). It can naturally infect many herbaceous and some woody plants and infect more than 430 plant species including several vegetable and woody crops in over 51 dicotyledonous families (Parrella et al., 2011). Potato is one of the most common hosts infected with AMV, which can cause various symptoms including mosaic, mottling and malformations, (Mughal et al., 2003; Bailiss and Ollenu, 1986). The yield of potatoes infected with AMV showing calico mosaic symptoms is reduced by about 20% (Miczynski and Hiruki, 1987).
In Egypt, AMV is considered as one of the most frequent viruses infecting potato in different locations (Gamal El-Din et al., 1994; El-Helaly et al., 2012).
This study was designed to genetically compare between AMV isolate infecting potato crop in Assiut governorate and other Egyptian and worldwide AMV isolates, estimate the genetic variability among AMV-Assiut isolate and worldwide isolates and also determine the genetic relationship among AMV-Assiut isolate and other Egyptian isolates as well as AMV worldwide isolates. These information will help to increase our understanding about AMV movement from country to country and mutation rate in virus genome and should put in consideration in any attempt to design along management strategy of this viral pathogen in Egypt.
MATERIALS AND METHODS
Source of viruses: Potato plant growing in experimental farm, Faculty of Agriculture, Assiut University showing typical AMV like symptoms including mosaic, mottling, malformation of leave and stunting were serologically tested and reacted positively with specific antibodies against AMV. The RNA extracted from these plants according to Ali et al. (2012) was tested in reverse transcriptase poly chain reaction using specific primer to amplify coat protein gene of AMV as described by Abdalla and Ali (2012a).
Sequencing: Sequencing was carried out in both directions using Big-Dye terminator cycle sequencing according to Sanger et al. (1977) at the core facility of Molecular Biology Unit, Assiut University, Assiut, Egypt using a sequencing instrument DNA Sequencing Applied Biosystem.
Phylogenetic analysis: Neighbor joining trees were generated with bootstrap 1000 from both nucleotide and amino acid sequence of AMV-Assiut isolate and worldwide AMV isolates available in the GenBank database from other geographical locations (Table 1) using the MEGA 5.02 program (Tamura et al., 2011).
RESULTS
Comparison between nucleotide sequences of AMV isolates from Assiut and worldwide isolates: Neighbor-joining tree generated from 20 nt sequences of Assuit-AMV and worldwide AMV isolates (Fig. 1), showed that AMV isolates clustered into two main groups (group I and II) and Egyptian isolates fall in both of these groups. Assiut-AMV isolate fall in group II with another Egyptian isolate from Wady Elnatron (Accession number: HG315522), while another Egyptian isolate from Elmonyfeya governorate fall into group I. The majority of AMV isolates in group I originated from countries in North, South America, Europe and Australia including USA, France, Italy, Canada, Chile, Mexico and Brazil, beside AMV isolate from Elmonyfeya-Egypt. While the majority of isolates in group II originated from Asian countries including China, Korea and Japan, beside isolates form Egypt (Assiut and Wady Elnatron).
| Table 1: | Alfalfa mosaic virus (AMV) worldwide isolates in GeneBank data base (used to compare coat protein gene of AMV-Assuit isolate and worldwide isolates |
The degree of variation in nucleotide sequences of CP genes of AMV isolates (Table 2) was up to 10%. Assiut-AMV isolate shared highest similarity in CP gene with isolated from Egypt (Wady Elnatron), followed by AMV isolates form China and Egyptian isolates form Elmonyfeya, as the degree of variation were only 4,8, 5.1 and 5.6%, respectively (Table 2). While, Assiut-AMV isolate shared the less degree of similarity in nucleotide sequences of CP gene with isolates from Brazil then isolates from Australia, as the degree of variation were 9.9 and 7.5%, respectively (Table 2).
Comparison between amino acid sequences of AMV isolates from Assiut and worldwide isolates: Neighbor-Joining (NJ) tree generated from 20 aa sequences of CP genes of Assuit-AMV isolate and worldwide isolates, showed that AMV isolates also formed two clusters, with additional clustering of AMV isolates in each groups into two separate subgroups (Fig. 2).
| Fig. 1: | Neighbor joining tree construct from amino acid sequences of AMV-CP gen of AMV isolate from Egypt and 19 worldwide AMV isolates |
| Table 2: | Pairwise distance among nucleotide sequences in coat protein gene of Alfalfa mosaic virus isolates from Assiut (Egypt) and worldwide AMV isolates |
But the difference between NJ tree constructed from nucleotide sequences and that one generated from amino acid sequences was the affiliation of each isolates in each groups, as some isolates which fall in group I in case of nucleotide tree fall into group II in case of amino acid tree. Egyptian isolates also fall in both groups. However, the affiliation of the isolates in groups was different than nucleotide tree.
| Fig. 2: | Neighbor joining tree construct from amino acid sequences of AMV-CP gen of AMV isolate from Egypt and 19 worldwide AMV isolates |
| Table 3: | Pairwise distance among amino acid sequences in coat protein gene of Alfalfa mosaic virus isolates from Assiut (Egypt) and worldwide AMV isolates |
Two Egyptian isolates (from Assiut and Elmonyfeya) fall in group I, but in different subgroups Ia and Ib, respectively (Fig. 2). While, Egyptian isolate (Wady Entron) fall into group II. The degree of variation in CP amino acid sequences of AMV isolates (Table 3) was less than degree of variation in nucleotide sequences and it was up to 4.3% (Table 3).
Assiut-AMV isolate shared highest similarity in aa sequences of CP gene with isolates from Croatia and Chile followed by AMV isolate form Egypt (Wady Elnatron), as the degree of variation which were only 2.1, 2.1 and 2.4%, respectively (Table 3).
| Fig. 3: | Neighbor joining tree construct from nucleotide sequences of AMV-CP genes of AMV isolate from worldwide AMV isolates to study the effect of host factor on virus divergence |
While, Assiut-AMV isolate shared the less degree of similarity in aa sequences of CP gene with isolates from Japan followed by an isolate from Brazil, as the degree of variation were 4.3 and 3%, respectively (Table 3).
Effect of host and geographic origin on relationship among AMV isolates: Results revealed that AMV has a wide host range and to study whether, host and geographical origin of each isolate has any effect on variation, a neighbor joining tree was constructed from CP gene 50 AMV worldwide isolates.
The NJ tree (Fig. 3), showed that AMV clustered into two main groups, with additional clustering of AMV in each groups into subgroups (a and b). Phylogenetic analysis showed neither clear structure among AMV isolates according to their geographical origin nor to their host origin. However, each subgroup contained isolates belong to different and far geographic origin and different host. The AMV-Assiut showed tendency to cluster according to geographic and host origin, as AMV-Assiut which was closely related to an isolate from Egypt (Wady Elnatron) and both isolates were originated from potato plants, supporting the close relation between these isolates, but the same subgroup also included isolates from different countries and different hosts, This fact contradict with the hypothesis of presence clear AMV structure according to geographic or host origin.
DISCUSSION
Alfalfa mosaic virus (AMV) is one of the most important viruses worldwide and has a very wide host range (Parrella et al., 2011; Jaspars and Bos, 1980). It causes severe disease in potatoes (Bailiss and Ollenu, 1986; Miczynski and Hiruki, 1987). In Egypt, AMV appeared on potato plants in several location causing severe lose (El-Helaly et al., 2012). Information about degree of variation in AMV and comparison among AMV strains from other countries will lead to better understanding of the similarities and differences among AMV isolates and will help to design a proper approach for detection and management of this virus (Xu and Nie, 2006).
An AMV isolate infecting potato crop in Assiut governorate (AMV-Assiut) was isolated and identified. Sequencing of CP gene of AMV-Assiut isolate showed that this isolate shared high degree of variability with other AMV isolates, this variability ranged from 4.8-10% in case of AMV isolate form Egypt (Wade Elnatron) and isolate from Brazil, respectively.
This degree of variation was similar to the degree of variability reported before by Al-Saleh and Amer (2013), who mentioned that AMV isolates from Saudi-Arabia shared similarity ranged from 90.3-99.3% with AMV worldwide isolates and Xu and Nie (2006), who revealed that the level of variation in CP among AMV isolates was up to 7% in amino acid sequence and up to 10% in nucleotide sequences. Moreover these data was in agreement with Parrella et al. (2010), who showed that variation in CP genes of AMV worldwide isolates was up to 7% at the nucleotide level. While Parrella et al. (2011) mentioned that the percentage of diversity in CP gene of AMV isolates ranged between 4.0-6.1 and 4.3-5.5% in nucleotide and amino acid sequences, respectively.
This high degree of variation in AMV isolates was reported before as AMV is one of the most biologically variable plant viruses with numerous natural variants having different pathogenicity (Crill et al., 1970; Massumi and Pour, 2010; Hajimorad and Francki, 1988).
This level of variation in AMV isolates was similar to degree of variation reported in other RNA viruses like Papaya ring spot virus, which was up to 12% at nt sequence (Abdalla and Ali, 2012b), but is still lower than nt variation reported in other RNA like Yam mosaic virus (YMV) (Bousalem et al., 2000) and Rice yellow mottle virus (RYMV) (Pinel et al., 2000), which showed nt variation in CP up to 28 and 22.4%, respectively.
The higher variation is character of all RMV viruses which are characterized by rapid mutation and evolution rate (Domingo and Holland, 1997). High variation in CP gen may suggest that coat protein may not the major gene responsible for symptoms se induced by AMV (Van der Vossen et al., 1996).
The degree of variation in amino acid sequences among AMV isolates was less than degree of variation in nucleotide sequences, as AMV-Assiut isolate shared a degree of variability ranged from 2.1-4.7% in case of AMV isolates from Croatia and Japan, respectively. This could be explained as some changes in nucleotide did not affect amino acid sequences (Wang et al., 1994). This agrees with other studies that reported this natural pressure is keeping virus primary structure as conserved as possible (Hema and Prasad, 2004).
The result of this study is in partial agreement with data reported before about AMV in Egypt by El-Helaly et al. (2012), who revealed that Egyptian isolate from Elmonyfeya governorate was closely related to other worldwide AMV isolates and showed similarity with other worldwide isolates which ranged from 97-98%. This difference in data may be due to that the current study used bigger number of isolates than number of isolates which were used in the aforementioned analysis, in addition that may be AMV-Assiut isolate is more distinctive than AMV isolate from Elmonyfeya, However more sequences of AMV isolates from different regions in Egypt is still required to present clear idea about variation among AMV isolates in Egypt.
Phylogenetic analysis, showed that AMV worldwide isolates tend to cluster in two main groups, in case of neighbor joining trees generated from either nucleotide or amino acid sequences (with additional formation of two subgroups in each main group), this tendency may indicate to presence of two distinctive strains of AMV two pathway of evolution of AMV isolates. Similar results were mention before as by Parrella et al. (2000), who reported that the topology of the trees obtained with two methods was essentially showing that AMV isolates clustering in two monophyletic groups close clustering of Italian strains in subgroup I and French strains in subgroup. This data in partial agreement with finding published by Kraal (1975), who mentioned that AMV could by divided into three different groups on the basic of amino acid sequences and Xu and Nie (2006), who divided AMV isolates into four different groups.
Study the effect of geographic or host origin on AMV variation and relationship did not show clear relation between either geographic origin or host origin and clustering (relation) among AMV isolates. The AMV isolates from different geographic and host origin tended to cluster in the same subgroup. These data contradict with the hypothesis suggested before about the effect of geographic distinctiveness of evolutionary dynamics of these AMV strains (Parrella et al., 2000).
The current study confirmed the high degree of variation in CP gene among MV isolates. This degree of variation should put in consideration in any attempt to control AMV, especially in long term management strategy. The current result revealed that AMV isolates could be divided into two main groups and AMV isolates from Egypt fall into both groups, indicating the possible existence of two strains of AMV isolates in Egypt. Additional molecular and serological studies are required to present more information about AMV isolates infecting potato in Egypt.