Occurrence and molecular characterization of wheat streak mosaic virus in wheat in Serbia

Ana Vučurović1,2*, Ivana Stanković1, Katarina Zečević1, Branka Petrović1, Goran Delibašić1 and Branka Krstić1 1University of Belgrade, Faculty of Agriculture, Institute of Phytomedicine, Department of Phytopathology, Nemanjina 6, 11080 Belgrade, Serbia 2National Institute of Biology, Department of Biotechnology and Systems Biology, Večna pot 111, 1000 Ljubljana, Slovenia *Corresponding author: ana.vucurovic@agrif.bg.ac.rs; ana.vucurovic@nib.si Received: 29 September 2020 Accepted: 22 October 2020 SUMMARY


INTRODUCTION
Wheat is the most widely grown crop in the world, providing for 20% of daily protein requirements of 4.5 billion people (Lucas, 2012).In Serbia, wheat is grown on 577.499 ha, with an average yield of 4.4 t/ha (RZS, 2019).Concerning the harvest area, wheat is the second major crop in Serbia aft er corn (RZS, 2019).Most of Serbian wheat is produced in the Vojvodina province (southern part of Pannonian Plane), which has a favorable climate for winter wheat cultivation (Šeremešić et al., 2017;RZS, 2019).
Viruses may become limiting factors for successful wheat production and numerous viral diseases compromise wheat production worldwide.More than 50 viruses are currently known to infect wheat (Lapierre & Signore, 2004;Ordon et al., 2009).Viruses of wheat and other cereals can be divided into two major groups, regarding their transmission: soil-borne viruses vectored by the plasmodiophorid Polymyxa graminis, and viruses transmitted by insects or mites (Ordon et al., 2009).Two soil-borne viruses, the soilborne cereal mosaic virus (SBCMV) and barley yellow mosaic virus (BaYMV) (Roberts, 2014), three insect transmitted viruses, the barley yellow dwarf viruses (BYDVs), cereal yellow dwarf viruses (CYDVs) and wheat dwarf virus (WDV), and the mite-transmitted wheat streak mosaic virus (WSMV) (Ordon et al., 2009;Singh & Kundu, 2018;Mishchenko et al., 2019) are the most important viruses that cause serious wheat diseases.In recent years, there has been a signifi cant increase in the number and prevalence of wheat viruses, but what is most threatening is the increase in their economic importance (Seifers et al., 2008;Spaar et al., 2008;Mishchenko et al., 2019).Global climate change is predicted to cause a further increase in the incidence and importance of wheat viruses, especially of viruses transmitted by aphids (BYDV and CYDV), leafh oppers (WDV) or mites (WSMV) (Ordon et al., 2009;Trębicki et al., 2015).
WSMV, the type member of the Tritimovirus genus in the family Potyviridae, is one of the most widespread and harmful viruses of cereal crops (Brunt et al., 1996;Rabenstein et al., 2002;Burrows et al., 2009;Singh & Kundu, 2018;Mishchenko et al., 2019).Almost a century ago, the disease caused by WSMV was first observed in the Central Great Plains of the USA and described as "yellow mosaic" of winter wheat (McKinney, 1937;Hunger, 2010).The virus is widely distributed in major wheatgrowing regions of Eurasia and North America, but also in Mexico, Brazil, Argentina, Australia, and New Zealand (Stenger & French, 2009;Hadi et al., 2011;Navia et al., 2013).Under natural conditions, the virus is transmitted primarily by wheat curl mites (WCM, Aceria toshicella Keifer [1969]) and by seeds of infected plants to a lesser extent (Skoracka et al., 2014;Singh & Kundu, 2018).Even though seed transmission occurs at low rates from 0.2 to 0.5% (up to 1.5%) and is not important locally, it enables global spreading of the virus by international trade and exchange of germplasm ( Jones et al., 2005;Singh et al., 2018).The host range of WSMV includes wheat, oat, barley and maize, but also many other wild and grown members of the Poaceae family (French & Stenger, 2002;Dráb et al., 2014;Chalupníková et al., 2017;Singh & Kundu, 2017).Yield losses caused by WSMV are estimated at 1-2% annually (Appel et al., 2014), but they can vary greatly, ranging from 7-13% in Kansas (Atkinson & Grant, 1967) to over 83% in Australia (Lanoiselet et al., 2008) or even cause a complete crop failure (Stenger & French, 2009).Subsequent financial losses are substantial, and only in the Kansas State (USA) in 2017 they were estimated at 76.8 billion US $ (Kansas Wheat Commission, 2017 http://kswheat.com/growers/wheat-streak-mosaic-virus).In some countries, such as the Czech Republic, WMSV is considered as a re-emerging pathogen since its significance dramatically increased after almost 30 years of absence (Chalupníková et al., 2017).
Th e fi rst and most prominent disease symptoms usually appear on fi eld margins, closest to the source of vector mites (Singh et al., 2018).Young leaves exhibit parallel pale green and yellow stripe forming mosaic patterns which, in case of early (autumn) infection, may progress over the spring to stunting, yellowing, marginal necrosis and subsequently to poor tillering (Vacke et al., 1986;Chalupníková et al., 2017;Singh & Kundu, 2017;Singh et al., 2018).Early disease symptoms can be misleading and confused with nutritional disorder, damage caused by chemicals or environmental eff ects (Singh et al., 2018).
In Serbia, WMSV was found for the fi rst time in the 1960s (Šutić & Tošić, 1964(Šutić & Tošić, , 1966)).Th e following studies by Tošić (1971) confi rmed a signifi cant presence of WSMV in important wheat production areas, as well as the presence of mixed infections with brome mosaic virus (BMV).Apart from WSMV and BMV in Serbia, BYDV-PAV, BYDV-MAV, CYDV-RPV and WDV are also present in wheat fields (Šutić & Tošić, 1964(Šutić & Tošić, , 1966;;Krstić et al., 2018;Stanković et al., 2019).After these initial WMSV studies, no additional investigation was conducted although the results at that time suggested a significant impact and distribution of WMSV.Moreover, virus-like symptoms have been increasingly noticed in wheat crops in Serbia over the last few years.Therefore, the objectives of this study were to determine the presence of WSMV in wheat crops, evaluate its distribution in the country, and to determine the genetic relationship of Serbian WMSV isolates with those from other parts of the world.

Field survey -collection of plant samples
In the spring of 2019, winter wheat samples showing virus-like symptoms, including pale green and yellow parallel stripes followed by mild to severe leaf rolling and stunting of plants, were randomly collected from 10 crops at eight locations: Bački Brestovac, Inđija, Dolovo, Bački Maglić, Lugovo, Gibarac, Umka and Vršac.Aft er visual inspection, a total of 62 symptomatic plants of fi ve wheat cultivars, including Anapurna, Apache, Salasar, Foxyl, and Sobred, were collected.Th e samples were transported to the laboratory in hand-handled cooler and stored at -20°C until RNA extraction and RT-PCR analyses were performed.

Molecular detection of wheat viruses
In order to determine the presence of wheat viruses in collected samples, individual or multiplex reverse transcription (RT)-PCR assays were carried out using specific primers for the detection of nine most economically important wheat viruses: barley yellow dwarf virus-PAV, -MAV, -SGV, -RMV (BYDV-PAV, -MAV, -SGV, and -RMV), cereal yellow dwarf virus-RPV (CYDV-RPV), wheat spindle streak virus (WSSMV), WSMV, BMV, and soil-borne wheat mosaic virus (SBWMV) (Deb & Anderson, 2008;Тrzmiel et al., 2016).Total RNAs were extracted from 100 mg of freeze-dried leaves of the collected samples by the CTAB method (Li et al., 2008) and used as a template in individual RT-PCR for the detection of BMV or multiplex RT-PCR assay for simultaneous detection of other mentioned wheat viruses.The RT-PCRs were performed using the One-Step RT-PCR kit (Qiagen GmbH, Germany) and different sets of virus-specific primers (Table 1).RNA extracted from healthy wheat plants and RT-PCR mix with RNase free water served as negative controls in each RT-PCR reaction.
Th e RT-PCR reaction mixture included 5μl of 5x Qiagen OneStep RT-PCR buff er, 400 μM of each of the four dNTPs, 1 μl of RT-PCR enzyme mix (Omniscript Reverse Transcriptase, Sensiscript Reverse Transcriptase, and HotStar Taq DNA Polymerase), 0.6 μM of each viral sense and complementary sense primer, and 1 μl of extracted RNA in a fi nal volume of 25 μl.Multiplex RT-PCR reactions were performed in a thermal cycler (Applied Biosystems 2720) under the following conditions: reverse transcription was performed at 50°C for 30 min, followed by an initial PCR denaturation step at 95°C for 15 min, and 6 cycles of denaturation at 94°C for 30 s, annealing at 60°C for 30 s with the annealing temperature decreasing by 1°C in each successive step and extension at 72°C for 30 s. Th ese 6 cycles were followed by 30 cycles at 95°C for 30 s, 55°C for 1 min, 72°C for 30 s and fi nal extension at 72°C for 10 min.For BMV, the fi rst strand cDNAs were synthesized at 50°C for 30 min and terminated at 95°C for 15 min, and then PCR was carried out by performing 35 cycles at 94°C for 30 s, 55°C for 30 s and 72°C for 1 min, followed by fi nal extension at 72°C for 10 min.Amplifi ed products were separated by 1.5% agarose gel electrophoresis, stained with ethidium bromide, and visualized under a UV transilluminator.

Sequence analysis
Th e identity of four selected Serbian WSMV isolates (98-19, 99-19, 102-19 and 120-19), originating from diff erent locations, was further confi rmed by amplifi cation of the 750 bp PCR fragment containing the N-terminal and core region of the coat protein gene using the primer pair WS8166F (5´ GAG AGC AAT ACT GCG TGT ACG 3´) and WS8909R (5´ GCA TAA TGG CTC GAA GTG ATG 3´) (Kúdela et al., 2008).Th e components of the RT-PCR reactions were as previously described, while amplifi cations were performed in a thermal cycler under the following conditions: reverse transcription was performed at 50°C for 30 min, followed by an initial PCR denaturation step at 95°C for 15 min, and 30 cycles of denaturation at 94°C for 45 s, annealing at 53°C for 30 s, extension at 72°C for 1 min; and a fi nal extension at 72°C for 10 min.Th e size of the amplifi ed products was determined as described in the previous section.
Aft er purifi cation with the QIAquick PCR Purifi cation Kit (Qiagen), RT-PCR products of four selected isolates were sequenced directly in both directions, using the same primer pair as in RT-PCR, and deposited in GenBank (Table 2).Sequences of the Serbian WSMV isolates were compared with each other and with the WSMV sequences available in the GenBank database using BLAST algorithm (http://www.ncbi.nlm.nih.gov/BLAST/),ClustalW (Th ompson et al., 1994) and MEGAX soft ware (Kumar et al., 2018).A p-distance model was applied for nucleotide (nt) and deduced amino acid (aa) sequence analyses and the divergence of the sequences of WSMV isolates was calculated aft er trimming to the length of the shortest fragment.

Phylogenetic tree
A maximum-likelihood phylogenetic tree was constructed using four Serbian WSMV isolates obtained in this study and 53 WSMV sequences retrieved from GenBank from other parts of the world (Table 2).Th e best-fi tting model of nt substitution was investigated using MODELTEST implemented in MEGAX, and the Kimura 2-parameter model Gamma distributed (K2+G) was chosen.Th e reliability of the obtained tree was evaluated using the bootstrap method based on 1000 replicates, and bootstrap values <50% were omitted.Intra-and inter-group diversity values were calculated as the average genetic distance.Sequence of a closely related Tritimovirus, oat necrotic mottle virus (ONMV), GenBank accession number AY377938, was used as the outgroup sequence.

Symptoms observed in the fi eld
During the spring of 2019, typical virus-like symptoms were observed in winter wheat fi elds in Serbian most important wheat growing regions.Plants exhibited prominent yellow stripes progressing to a mosaic pattern on leaves (Figure 1), which later merged in chlorotic or yellow parts of leaves or whole leaves (Figure 2).Th e observed symptoms sometimes included mild to severe leaf deformations, such as leaf rolling and wilting.During the early spring, symptoms appeared on plants at fi eld edges.At the end of the growing season, a characteristic disease gradient from very severe on fi eld edges to decreasingly severe towards fi eld depth was observed in some locations, while in others infected plants were randomly distributed across the fi eld.Infected plants exhibited also stunting and reduction in tiller number.

Molecular detection of WSMV
Molecular analysis of wheat samples revealed the presence of WSMV in 58.1% of the tested samples collected from seven commercial wheat crops at fi ve locations: Bački Brestovac, Inđija, Bački Maglić, Lugovo, and Vršac (Table 3).Th ree locations, Dolovo, Gibarac and Umka, were proved to be free of either WSMV or any other tested virus.Th e highest incidence of WSMV (100% samples testing positive) was in the locations Inđija and Bački Maglić, while virus presence was proved in all three inspected crops at the location Bački Brestovac.At the location Vršac, WSMV was detected in 71.4%, while the virus was confi rmed in 62.5% of the tested samples at the Lugovo location.

Molecular identifi cation and phylogenetic analysis of WSMV
Primer pair WS8166F/WS8909R specifi cally amplifi ed fragments of the expected size of 750 bp in all four selected isolates.Th e amplifi ed fragments were sequenced and four sequences of Serbian WSMV isolates generated in this study were submitted to GenBank database of the NCBI and assigned with accession numbers shown in Table 2.
Th e CP gene sequences of the four Serbian isolates shared nt identities of 98.16% to 99.02% (99.57 to 100% aa identities).Th e highest percentage of nt identity was shown between the isolates 98-19 and 99-19 (99.02%), while 99-19 was the most distant from the isolate 102-19 (98.16%).BLAST search analysis revealed that the sequences of four Serbian WSMV isolates proved to be identical at the nucleotide level from 98.59 to 99.44% with those from other parts of the world.Nucleotide sequences of the isolates 98-19 and 99-19 showed the highest homology with a Polish barley isolate (MH939146) of 99.44% and 99.02%, respectively.Sequences of the isolates 102-19 and 120-19 had the highest homology with a Czech wheat isolate (FJ216409) of 98.59% and 99.28%, respectively.
All four Serbian WSMV isolates had a specifi c deletion of three nucleotides at the position 8412-8414 nt in the CP gene.Positions are numbered according to the positions of the WSMV reference isolate (NC_001886).Th is deletion of triplet codon GCA resulted in the deletion of glycine amino acid at the position 2761 (Figure 3).A Maximum Likelihood tree (Figure 4) was constructed using a 683 nt fragment of the CP gene from 57 WSMV isolates from all over the world.Th e phylogenetic tree indicated a division of WSMV population in fi ve clades (Clades A, B, B1, C and D) with high bootstrap values for major clades containing more than one isolate B, B1 and D (84, 100 and 93, respectively).Th e overall genetic diversity of WSMV sequences in the reconstructed phylogenetic tree was 0.083±0.006.Genetic diversities between clades ranged from 0.085±0.010to 0.3286±0.017.Clade A consisted of a single isolate from Mexico (AF285170), which is genetically most distant from the other WSMV.Clade B consisted of Eurasian wheat isolates from 13 diff erent countries (Iran, Turkey, Czeck Republic, Germany, Slovakia, Serbia, Hungary, Lithuania, Ukraine, Russia, France, Poland and Austria).All four Serbian isolates were grouped together with most of the isolates within clade B originating from Europe and Asia.Serbian WSMV isolates clustered closely with isolates from the Czech Republic, Slovakia, Hungary and Turkey.Clade B showed the greatest intragroup variability in the phylogenetic tree (0.029±0.002) and isolates of this group are characterized by deletion of a triplet codon GCA (Glycine amino acid) in the CP gene sequence.Clade B1 consisted of three isolates from grasses collected in the Check Republic and was most closely related to clade B, showing 0.1134±0.011inter-group variability.Clade C consisted of a single isolate from Iran.Th e most divergent clade, considering geographical distribution, was clade D, comprising isolates from fi ve continents (Europe, Asia, Australia, North and South America), but intra-group sequence diversity in this clade was slightly lower than in clade B (0.026±0.003).

DISCUSSION
Although wheat is the second most important food crop in Serbia and despite the fact that Tošić (1971) reported the presence and signifi cant distribution of WSMV in important wheat growing areas in Serbia, no data were available over the past decades on the presence and distribution of WSMV or other wheat infecting viruses in Serbia.Th erefore, the occurrence, incidence and prevalence of wheat viruses in Serbia are unknown today.Only recently, a survey searching for wheat viruses, initiated by Stanković et al. (2019), showed that WSMV is present and widespread in the country.Today, WSMV is the most common wheat virus around the world which causes losses of up to 100% (Stenger & French, 2009;Hadi et al., 2011;Navia et al., 2013).Yield losses caused by WSMV can reach up to 464.5 US $ per hectare (Velandia et al., 2010), endangering production or even making it entirely unsustainable.WSMV aff ects not only yield, but also root development and water use effi ciency of infected wheat plants (Price et al., 2010).In recent years, WSMV has become a re-emerging virus in cereal crops in the Czech Republic (Chalupníková et al., 2017).
Th is study showed that WSMV occurred as a single infection in fi ve out of eight inspected wheat growing locations in Serbia during 2019.Th e presence of WSMV was detected in 58.1% of the tested samples and all samples were negative for the presence of other tested viruses.All collected samples originating from three locations (Inđija, Bački Maglić and Bački Brestovac) were WSMV positive, and a lower but signifi cant percentage of samples from two other locations (Vršac and Lugovo) were also positive (71.4% and 62.5%, respectively).WSMV commonly occurs in complexes with other wheat viruses (Byamukama et al., 2013).Th e investigation carried out by Tošić (1971) revealed also the presence of mixed infection of WSMV and BMV, but in our present study WSMV was found only as single infection.Since none of the tested viruses, including WSMV, were detected in three wheat fi elds, further investigation will be focusing on identifying the causal agent(s).
Early-season symptoms mostly appeared on plants at fi eld margins, and as the season progressed the infected plants developed an obvious disease gradient.In some fi elds, where the presence of WSMV was not proved, symptomatic plants were scattered throughout the fi eld.Chalupníková et al. ( 2017) observed a similar distribution of WSMV symptoms in the Czech Republic.Symptoms of streak mosaic and yellowing decreased in severity towards fi eld centre, as noticed also by Workneh et al. (2009).Migration of the wheat curl mite vector, Aceria tosichella, from grassy areas and bordering crops into wheat fi elds, stimulates the spreading of infection (Hunger, 2010).
A comparison of nucleotide sequences of WSMV isolates collected from diff erent wheat-growing regions of the world has confi rmed the existence of WSMV genetic diversity (Singh et al., 2018).Based on the CP gene sequence, the WSMV population is divided into four clades, named A, B, C, and D, and a recently introduced clade B1 (Stenger & French, 2009;Robinson & Murray, 2013;Singh & Kundu, 2017;Singh et al., 2018;Mishchenko et al., 2019).Topology of the phylogenetic tree obtained in this study and nucleotide similarities between clades are in accordance with previous studies (Stenger & French, 2009;Robinson & Murray, 2013;Singh & Kundu, 2017;Singh et al., 2018;Mishchenko et al., 2019).All Serbian WSMV isolates were grouped into clade B together with other European WSMV isolates and one isolate from Iran, implying that they share a single common ancestor.Th e analysis also indicated the existence of a single genotype of WSMV in Serbia.Moreover, nucleotide sequences of the CP gene of Serbian WSMV isolates are characterized by a deleted triplet codon GCA at nucleotide position 8412-8414, resulting in deletion of the amino acid glycine (Gly 2761 ), as previously reported for isolates belonging to B and B1 clades (Gadiou et al., 2009;Mishchenko et al., 2019).All these results imply that European isolates are common and had been widely dispersed throughout European countries from a single focus (Gadiou et al., 2009).Unlike the genetic uniformity of WSMV isolates in Europe, considerable genetic variation in WSMV populations was found in the USA (Robinson & Murray, 2013).Reports on three genotypes of WSMV coexisting in Iran (Schubert et al., 2015), and discovery of European WSMV isolates in the U.S. Pacifi c Northwest region (Robinson & Murray, 2013) and Canada (Bennypaul et al., 2019), have revealed transferring and diverse distribution of WSMV globally.Th e observed WSMV diversity in the USA and obvious introduction by movement of viruliferous vectors or infected seed through continually expending trade in plants and plant products, indicate a constant need to study and evaluate WSMV population structure.
Th is study provides the fi rst information on the presence of WSMV in Serbia aft er initial investigation that was carried out almost 50 years ago.WSMV continues to be a threat to wheat production in Serbia and its importance may increase in the future.Further investigation is needed to provide information on the biology, ecology and epidemiology of the disease and its vector.Considering that a substantial number (41.9%) of collected samples during the survey were negative for eight tested viruses, a further thorough survey and testing for the presence of other wheat-infecting viruses are required.In addition, the results of this work provide the fi rst data on molecular characterisation of WSMV isolates originating from Serbia, indicating their close relationship with other European isolates and existence of a single genotype in the country.The extent of a possibly greater genetic diversity of WSMV in Serbia will be assessed when more isolates of diff erent origin have been collected.

Figure 1 .Figure 2 .
Figure 1.Wheat streak mosaic virus: parallel mosaic and stripes on wheat leaves

Figure 3 .Figure 4 .
Figure 3. Comparative analysis of a part of amino acid sequences of the coat protein (CP) gene of Serbian wheat streak mosaic virus isolates and referent wheat streak mosaic virus isolate (NC_001886) showing deletion of glycine aa residue at position 2761

Table 2 .
Coat protein gene sequences of wheat streak mosaic virus isolates used for phylogenetic analyses

Table 2 -
continued.Coat protein gene sequences of wheat streak mosaic virus isolates used for phylogenetic analyses

Table 3 .
Number of tested and percentage of wheat streak mosaic virus positive samples in 2019