Journal of Integrative Agriculture 2017, 16(0): Available online at ScienceDirect

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1 Journal of Integrative Agriculture 2017, 16(0): Available online at ScienceDirect RESEARCH ARTICLE Wheat streak mosaic virus: incidence in field crops, potential reservoir within grass species and uptake in winter wheat cultivars Jana Chalupniková 1, 2, Jiban Kumar Kundu 1, Khushwant Singh 1, Pavla Bartaková 1, Eva Beoni 1 1 Division of Crop Protection and Plant Health, Crop Research Institute, Drnovská 507, Prague, Czech Republic 2 Department of Experimental Plant Biology, Faculty of Science, Charles University in Prague, Albertov 6, Prague, Czech Republic Abstract Wheat streak mosaic virus (WSMV) has become a re-emerging pathogen in cereal crops in the Czech Republic. WSMV was first reported in the former Czechoslovakia in the early 1980s, and then no record of the virus was documented until The incidence of the virus was recorded in recent years in several winter wheat field and many grass species. Here, we surveyed the incidence of WSMV in cereal crops. The results demonstrated the existence of the virus in winter wheat and volunteer wheat during each year of the monitoring period, which spanned from Although the range of infected samples was low (6.4% of the total tested samples), a high incidence of well-distributed virus was recorded. In at least six fields, the virus reached severe and potentially epidemic levels. In accordance with our previous report detailing WSMV infection of native grasses, we tested several grass species commonly grown in the Czech Republic. We found that some grass species acted as experimental hosts and possible reservoirs of the virus; these included Anthoxanthum odoratum (sweet vernal grass), Arrhenatherum elatius (false oat-grass), Lolium multiflorum (Italian rye-grass), Bromus japonicus (Japanese chess), Echinochloa crus-galli (barnyard grass), Holcus lanatus (meadow soft grass) and Holcus mollis (creeping soft grass). Some of these grass species are also important weeds of cereals, which may be the potential source of WSMV infection in cereal crops. Several widely used winter wheat cultivars were tested in the field after artificial inoculation with WSMV to evaluate virus titre by RT-qPCR. Overall, the tested cultivars had a low virus titre, which is associated with mild disease symptoms and may provide a good level of crop resistance to WSMV. Keywords: WSMV, survey, grass species, cereal crops 1. Introduction Received 14 July, 2016 Accepted 19 September, 2016 Correspondence Jiban Kumar Kundu, Tel: , Fax: , jiban@vurv.cz 2017, CAAS. All rights reserved. Published by Elsevier Ltd. doi: /S (16) Wheat streak mosaic virus (WSMV) is the type member of the genus Tritimovirus of the family Potyviridae (Rabenstein et al. 2004). WSMV is widespread throughout the world and represents a severe threat in most wheat-growing regions (Rabenstein et al. 2002; Hadi et al. 2011; Coutts et al. 2014). WSMV infects many plant species of the family Poaceae (Brakke 1971; French and Stenger 2002), including wheat (Triticum aestivum L.), oat (Avena sativa L.), barley (Hor-

2 *** et al. Journal of Integrative Agriculture 2017, 16(0): deum vulgare L.), maize (Zea mays L.), millet (Panicum, Setaria, and Echinochloa spp.) and several grasses (see Table 1). Symptoms caused by WSMV include leaf mottling (mosaic pattern of green and chlorotic zones) and leaf streaking and are mainly observed in wheat (Vacke et al. 1986; Murray et al. 1998). The symptoms may progress to chlorosis and severe stunting of the plant, and in many cases, plants become sterile or produce shrivelled seeds (Ellis et al. 2004). WSMV is transmitted by wheat curl mites (WCMs, Aceria tosichella) (Slykhuis 1955; Orlob 1966), which are dispersed passively by air currents (Slykhuis 1955). The virus is also transmitted at low levels by seeds in wheat (Jones et al. 2005). Estimated levels of WSMV in seed lots originating from infected commercial wheat crops range from 0 to 0.22%, with an overall transmission rate of 0.06% (Lanoiselet et al. 2008). WSMV was first reported in the Great Plains Region (GPR) of North America in Since then, WSMV has been recorded in many wheat-growing regions of the world, including North and South America, Europe, the Middle East, Asia, Australia and New Zealand (Navia et al. 2013; Skoasracka et al. 2014). Crop losses caused by WSMV vary widely, ranging from 7 to 13% in Kansas (Atkinson and Grant 1967), reaching 18% in Canada (Christian and Willis 1993) and reaching up to 83% in wheat in Australia (Lanoiselet et al. 2008). WSMV spreads to cereal crops from virus reservoirs, which can include volunteer wheat plants and some grasses. At least 45 grass species are reported to be natural hosts for WSMV, and the majority of them are annuals (Sill and Agusiobo 1955; Christian and Willis 1993; French and Stenger 2002; Ito et al. 2012). Many of these WSMV grass hosts are also hosts for WCM (for review, see Navia et al. 2013). There is limited protection of cereal crops against WSMV and its WCM vector. Host resistance against the virus and/ or vector is the most effective way to reduce the yield loss caused by WSMV (Thomas et al. 2004; Richardson et al. 2014). Three resistance genes, known as Wsm1, Wsm2, and Wsm3, have been identified and introduced in wheat breeding lines. Wsm1 and Wsm2 resistance sources were found to be more temperature-sensitive (Seifers et al. 2013) than Wsm3 (Fahim et al. 2012); however, they have not yet been introduced into a commercial cultivar. The Wsm2 gene is the most widely used and has been successfully introduced into several wheat cultivars, including RonL (Seifers et al. 2006), Snowmass (Haley et al. 2011), Clara CL (Martin et al. 2014) and Oakley CL (Zhang et al. 2015a). However, with the deployment of WSMV-resistant cultivars, the limited resistance sources in these cultivars may be broken down by selective pressure on the virus (Zhang et al. 2015b). WSMV was first reported in the former Czechoslovakia in the early 1980s (Vacke et al. 1986). The incidence of the virus in recent years has become more frequent in winter wheat crops (Gadiou et al. 2009; Dráb et al. 2015). Vacke et al. (1986) reported the existence of WSMV in wheat, barley, oat and some cultivars of corn as well as in some grass species, including Avena fatua, Avena strigesa, Panicum miliaceum, Lolium multiflorum, Lagurus ovatus and Digitaria sanguinalis. In the present work, we report the results of a four-year survey of the incidence of WSMV in cereal crops, which was recorded for several winter wheat fields each year. We previously found that several grass species can become naturally infected with WSMV (Dráb et al. 2014), which may be a source of virus inoculum for cereal crops. We tested several grass species commonly grown in the Czech Republic and found that some of them were experimental hosts and possible reservoirs of the virus, such as Anthoxanthum odoratum (sweet vernal grass), Arrhenatherum elatius (false oat-grass), L. multiflorum (Italian rye-grass), Bromus japonicus (Japanese chess), Echinochloa crus-galli (barnyard grass), Holcus lanatus (meadow soft grass) and Holcus mollis (creeping soft grass). Several widely used winter wheat cultivars were tested in field experiments after artificial inoculation with WSMV to evaluate virus titre using real-time RT-qPCR. Several of the wheat cultivars, including Caldwell, Cubus, Florida, Matchball and Cim rana, had low levels of virus uptake. Furthermore, in one triticale cultivar, Koler, WSMV was undetected even when highly sensitive RT-qPCR was used. This low virus uptake in cultivars may be associated with a significant level of crop resistance to WSMV. 2. Materials and methods 2.1. Survey of WSMV in crop and grass hosts Samples from cereal fields, the fields margins and perennial meadows were collected from in different regions of the Czech Republic. The samples were collected during the early spring to autumn period randomly based on disease suspicion in the fields (Table 2) Experimental hosts within grass species Twenty-four species of grass (see Table 2) from Poaceae that naturally occur in the agro-ecosystem of the Czech Republic were tested. The plants were grown in greenhouses, and they were mechanically inoculated with a WSMV isolate (CZlab, accession number FJ216408) at the two-leaf stage. To accomplish this, individual plant leaves were smeared with sap containing the WSMV isolate with 0.1 mol L 1 phosphate buffer (ph 7.2) and carborundum powder. One month post-inoculation, the plants were tested by TAS-ELISA and then by RT-PCR as described in Gadiou et al. (2009).

3 4 *** et al. Journal of Integrative Agriculture 2017, 16(0): Table 1 Host species of Wheat streak mosaic virus (WSMV) Host Common name References Agropyron repens Couch grass Dráb et al Agrostis capillaris Common Bent In this study Alopecurus pratensis Meadow foxtail Dráb et al Anthoxanthum odoratum Sweet vernal-grass In this study Arrhenatherum elatius False oat-grass Dráb et al Austrostipa compressa Speargrass Vincent et al Aegilops cylindrica Jointed goatgrass Sill et al Avena barbata Bearded oat Coutts et al Avena fatua Wild oat Vacke et al Avena sativa Oat Brakke 1971 Avena strigesa Wild oats Vacke et al Avena sterilis Wild oats Murray et al Briza maxima Blowfly grass Coutts et al Bromus arvenis Field brone Sill et al Bromus diandrus Great brome Murray et al Bromus japonicus Japanese brome Wegulo et al Bromus rigidus Brome grass Coutts et al Bromus secalinus Cheat grass Sill et al Bromus tectorum Downy brome Sill et al Cynodon dactylon Couch grass Ellis et al Cenchrus longispinus Mat sandbur Connin 1956 Cenchrus pauciflours Sandbur Wegulo et al Digitaria sanguinalis Hairy crab grass Vacke et al. 1986; Somensen and Sill 1970 Echinochloa crus-galli Barnyardgrass Sill and Connin 1953 Echinochloa colonum Junglerice Khadivar and Nasrolahnejad 2009 Elymus repens Quackgrass Ito et al Eragrostis cilianensis Stink grass Connin 1956 Eragrostis curvula African lovegrass Ellis et al Eriochloa acuminata Tapertip cupgrass Seifers et al Eriochloa contracta Prairie cupgrass Christian and Willis 1993 Eleusine indica Crowsfoot Murray et al Eleusine tristachya Spike goosegrass Ellis et al Elymus canadensis Canada wild rye Ito et al Holcus lanatus Soft-grass In this study Holcus mollis Creeping soft grass In this study Hordeum leporinum Barley grass Coutts et al Hordeum vulgare Barley Brakke 1971 Lagurus ovatus Hare's-tail Vacke et al Lolium mitiflorum Annual ryegrass Vacke et al. 1986; Ellis et al Lolium rigidum Ryegrass Murray et al. 2005; Coutts et al Panicum dichotomiflorum Fall panicgrass Sill and Connin 1953 Panicum capillare Witch grass Coutts et al. 2008a, b Panicum millaceum Broomcorn millet Sill and Agusiobo 1955; Vacke et al. 1986; Ellis et al Pennisetum glaucum Pearl millet Seifers et al Phalaris aquatica phalaris Ellis et al Phleum pratense Timothy-grass Dráb et al Poa pratensis Bluegrass Ito et al. 2012; Dráb et al Secale cereale Cereal rye Vacke et al. 1986; Ito et al Setaria verticellata Whorled pigeon grass Murray et al Setaria viridis Green bristlegrass Sill and Connin 1953 Setaria italica Foxtail millet Truol et al Sorghum bicolor Sorgum Seifers et al Tragus australianus Small burr grass Coutts et al. 2008a, b Triticum aestivum Wheat Brakke 1971 Zea mays Maize Brakke 1971

4 *** et al. Journal of Integrative Agriculture 2017, 16(0): Table 2 Survey of the incidence of WSMV in wheat and barley crops and volunteer plants Sampling year Crop species Total tested samples Number of sampling fields Number of WSMV-positive samples Number of MSMV-infected field Percentage of WSMV-infected samples (%) Percentage of WSMV-infected fields (%) 2013 Winter wheat Winter barley Spring barley Winter wheat Winter barley Winter wheat Winter barley Spring barley Wheat volunteer Winter wheat Winter barley Spring barley Total Detection of WSMV TAS-ELISA WSMV was detected by triple anitbody sandwich (TAS)-ELISA using commercial antiserum (SEDIAG SAS, France) according to the manufacturer s instructions. RNA isolation WSMV-infected leaves were used for total RNA extraction using a Spectrum Plant Total RNA Kit (Sigma Aldrich, USA) following the manufacturer s instructions with minor modifications. A total of 100 mg of infected leaves was homogenized in liquid nitrogen to obtain a very fine powder. 500 µl of lysis buffer supplemented with 2-mercaptoethanol were added and mixed vigorously. The samples were incubated at 60 C for 5 min and centrifuged at r min 1 for 5 min. The supernatant was transferred to a filtration column and centrifuged again. 500 µl of binding solution were added to the filtrate, followed by centrifugation for 1 min at r min 1. The column was washed with 500 μl of wash buffer I and centrifuged. The column was washed three times with 500 μl of wash buffer II before adding 50 μl of elution buffer. The RNA was quantified using a Nanodrop 2000 (Thermo Scientific, USA) and stored at 80 C for further use. RNA was treated with DNase I using DNA-free (Ambion, USA) RT-PCR cdna synthesis First-strand cdna was synthesised using a Reverse Transcription System (Promega, USA). A reaction mixture composed of 1 µg RNA and 0.5 µg random primer was incubated at 70 C for 5 min and chilled on ice for 2 min. Then, the following components were added in the given order: 5 reaction buffer, 20 U µl 1 RNasin RNase inhibitor, 10 mmol L 1 dntp mix and 200 U µl 1 M-MLV reverse transcriptase enzyme. The mixture was incubated at 37 C for 1 h. The reaction was stopped by heating at 70 C for 10 min and subsequent chilling on ice. PCR cdna was amplified as described in Gadiou nt et al. (2009) using the primer pair WSMspeFw (5 - GCCTCGACACGGGAGCTA nt 3 ) and WSMspeRv ( nt ACCCATCCAGGAAGCAAGG nt -3 ). The reaction mixture contained cdna, 10 μmol L 1 each of forward and reverse primers, and 2 DreamTaq Green PCR Master Mix (Thermo Scientific, USA). The PCR conditions were as follows: initial denaturation at 94 C for 5 min, followed by 30 amplification cycles (94 C, 45 s; 66 C, 30 s; and 72 C, 1 min), and a final extension at 72 C for 10 min. The PCR assay yielded a 358-bp product. The PCR product was then separated on a 1% agarose gel and stained with SYBR Safe DNA Gel Stain (Invitrogen, USA) Quantitative analysis of WSMV in winter wheat cultivars Plant material and field experiment Quantitative analysis of WSMV was performed in wheat cultivars, including Florida, Vlada, Alcedo, Caldwell, Rocky, Ludwig, Cubus, Manager, Muszelka, Cim rana, Tobak, Matchball, Genius, Asano, and Bohemia as well as in the triticale cultivar Kolor. These cultivars are commonly grown in the Czech Republic. Each cultivar was grown in two rows (total ten plants per row) and in two replicates, one for virus inoculation and another for a non-inoculated control. The plants were mechanically inoculated with a WSMV isolate (CZlab, accession number FJ216408) at the three-leaf stage during autumn as described above. The leaf samples of the tested cultivars were collected in the spring at Feekes growth stage 9 for WSMV titre analysis by RT-qPCR. RT-qPCR RNA was isolated as described above. qpcr was performed as described in Dráb et al. (2014) using primer pair WSMV-F ( nt AAGTGCAGAACAGCGTTG 9

5 6 *** et al. Journal of Integrative Agriculture 2017, 16(0): nt -3 )/WSMV-R ( nt AAACTGTGCGTGTTCTCC nt - 3 ) (Tatineni et al. 2010). A primer pair for wheat elongation factor 1α (eef1α) served as a positive control, enabling the assessment of RNA quality (Jarošová and Kundu 2010). The qpcr reaction was performed on a LightCycler 480 Real-Time PCR System (Switzerland). A 250 nmol L 1 primer concentration was used in the qpcr reaction mixture, and the primer annealing temperature was 60 C. The total cdna concentration in the reaction mixture was 3 ng ml 1. The amplification protocol was as follows: denaturation at 95 C for 10 min, followed by 40 cycles of 95 C for 5 s, 60 C for 30 s and 72 C for 10 s. Finally, melting analysis of the PCR products was performed. qpcr data analysis The titre of WSMV in the tested samples was calculated according to a ten-fold standard dilution curve constructed using the PCR product of a positive WSMV sample. Calculations were made using LightCycler 480 Real-Time PCR System software. The PCR product quantity in µg was converted to pmol using the average molecular weight of a ribonucleotide (340 Da) and the number of bases of the transcript (Nb). The following mathematical formula was applied: pmol of dsdna=μg of dsdna) 10 6 pg μg 1 /660 pmol pg -1 /Nb. Avogadro s constant ( molecules mol 1 ) was used to estimate the number of transcripts. A graphical representation of the data was constructed using the programme Microsoft Excel. 3. Results 3.1. Incidence of WSMV in crops were tested after mechanical inoculation with WSMV. The virus was detected in eight species, including Agrostis capillaris, A. odoratum, L. multiflorum, B. japonicas, A. elatius, E. crus-galli, H. lanatus and H. mollis (Table 3 and Fig. 2). WSMV was generally detectable by both TAS-ELISA and RT-PCR, except for in A. capillaris and H. lanatus, in which WSMV was detected solely by RT-PCR Differences in virus uptake among wheat cultivars WSMV uptake in commonly grown winter wheat cultivars was tested by analysis of virus titre by RT-qPCR. Ten cul- Fig. 1 Map of Wheat streak mosaic virus (WSMV) occurrence in the fields of the Czech Republic. WSMV infections are indicated as stars: black stars, fields with individual infected plants; red stars, fields with severe infections. WSMV incidence was surveyed from the years 2013 to Sampling was performed in a random manner in a total of 134 fields; the fields included winter wheat and barley, spring barley and volunteer wheat plants. In total, 876 samples were tested, and the infection rate varied among years and crops. WSMV was detected solely in winter wheat, in both crop and voluntary plants. The average rate of WS- MV-infected samples was 6.4% (56 samples) in total, and among the 94 wheat fields and four wheat volunteer fields tested, the virus was detected in 19 wheat fields and one wheat volunteer field (Table 2). These results indicate that only a few individual infected plants were detected in most of the fields. However, in six fields, the virus was found at a high incidence and was widely distributed; these included two fields sampled in 2013, one field sampled in 2015 and three fields sampled in 2016 (the locations of these fields are indicated in red on the map in Fig. 1) Experimental hosts within the grass species Nineteen commonly grown grass species, including weeds, Table 3 Detection of WSMV in grass species after artificial inoculation with WSMV Number of WSMV-positive plants/ Grass species Number of tested plants TAS-ELISA RT-PCR Aera spica-venti 0/2 0/2 A. capillaris 0/7 2/7 Agrostis stolonifera 0/7 0/7 A. odoratum 1/7 1/7 A. elatius 2/7 2/7 Bromus erectus 0/4 0/4 B. japonicus 4/7 4/7 Bromus sterilis 0/4 0/4 Cynosurus cristatus 0/7 0/7 Dactylis glomerata 0/5 0/5 E. crus-galli 5/7 5/7 Holcus lanatus 0/5 1/5 H. mollis 1/5 1/5 Hordeum murinum 0/5 0/5 L. multiflorum 2/6 2/6 P. pratense 0/7 0/7 Poa annua 0/5 0/5 Poa trivialis 0/6 0/6 Trisetum flavescens 0/7 0/7

6 *** et al. Journal of Integrative Agriculture 2017, 16(0): tivars were tested in field experiments, including a triticale cultivar. Significant differences in virus titre were detected in the tested cultivars (Fig. 3). The WSMV uptake levels that were recorded in the tested cultivars were grouped as follows: I) low: Caldwell, Cubus, Florida, Matchball and Cim rana; II) medium: Rocky, Vláda, Alcedo, Muszelka and Manager; and III) high: Ludwig, Bohemia, Asano, Tobak and Genius. Notably, WSMV was undetectable in the triticale cultivar Koler (Fig. 3). 4. Discussion The results of the current survey of the incidence of Wheat streak mosaic virus (WSMV) in the Czech Republic showed a low occurrence of the virus and demonstrated that the virus was present only in wheat. Winter wheat is the main host of WSMV and the most commonly cultivated cereal crop in the Czech Republic. In our study, the majority of tested samples came from wheat crops rather than from minor barley, in which WSMV was not detected (see Table 2). 358 bp M Fig. 2 Electrophoresis of RT-PCR products for the detection of WSMV in grass species after artificial inoculation with WSMV. SYBR Green-stained 1% agarose gel showing a PCR product of 358 bp. Lanes 1 19, A. spica-venti, A. odoratum, T. flavescens, L. multiflorum, D. glomerata, C. cristatus, B. japonicus, B. sterilis, B. erectus, A. elatius, A. pratensis, A. capillaris, A. stolonifera, E. crus-galli, P. annua, P. trivialis, H. lanatus, H. mollis, and H. murinum; lane M, GeneRuler 100 bp Plus DNA Ladder (Thermo Scientific, USA). We previously found that WSMV occurs very occasionally and that only individual plants within a field are affected in general. In at least six winter wheat fields (early sowing), we found a high incidence of WSMV, and the virus was spread throughout the fields (see the WSMV symptoms in wheat cultivars in Fig. 4-A nad B). These results suggest that there is significant potential for WSMV infection to increase in crops. The warmer autumns and winters experienced by Central Europe in recent years have created favourable preconditions for the life cycle of the virus vector, namely, wheat curl mites (WCMs). WCMs have been reported to survive for months at near-freezing temperatures, including for several days at approximately 18 C (Townsend and Johnson 1996). No report on the occurrence of WCMs in the Czech Republic is currently available, and several attempts to identify WCMs in infected wheat crops were unsuccessful in our hands. However, WCMs have been reported to be abundant in neighbouring countries (Skoracka et al. 2014). The incidence of WSMV in natural grass species (Dráb et al. 2014) indicates the presence of the vector in the agro-ecosystem and thereby the introduction of the virus into crops. Another possibility for WSMV introduction is through infected seed, which may have occurred at a low level; this type of dissemination of the virus was reported in New Zealand (Lebas et al. 2009). The transmission rate of WSMV via seed ranges up to 1.5% (Jones et al. 2005), and seeds are likely an important source of virus inoculum when WCMs are also present (Lanoiselet et al. 2008). WSMV may cause significant yield losses in wheat, which may reach over 90% (Hadi et al. 2011) depending on weather conditions, cultivars and growth stage affected by virus infection. Generally, infections during the early stages of plant growth result in higher yield losses (Hunger et al. 1992; Baley et al. 2001). WSMV infections are also associated with reductions in root biomass and the efficiency of water uptake (Price WSMV copy number 1.40E E E E E E E E+00 WSMV titre analysis by RT-qPCR Kolor Caldwell Cubus Florida Matchball Cim rana Rocky Vláda Alcedo Muszelka Manager Ludwig Bohemia Asano Tobak Genius Cultivar Fig. 3 WSMV titre (copy number) in wheat cultivars and triticale cultivar Koler. One sample from each cultivar was tested.

7 8 *** et al. Journal of Integrative Agriculture 2017, 16(0): A B Fig. 4 WSMV symptoms in a naturally infected field growing wheat cultivars Hymack (A) Bodyček (B). Symptoms appear as small chlorotic lines, yellow and green streaks and a mosaic pattern in the leaves. The symptomatic leaves later become necrotic and dried out. et al. 2010), creating serious concerns with regard to climate change. In addition to crops, grass species are major hosts for both WSMV and WCMs, and a good number of them are common hosts (reviewed in Navia et al. 2013). In the Czech Republic, we observed the natural occurrence of WSMV in several grass species, including E. repens, A. pratensis, A. elatius, P. pratense and P. pratensis (Dráb et al. 2014). Several other species, including A. fatua, D. sanguinalis, L. multiflorum and P. miliaceum, were also reported by Vacke et al. (1986). Hence, grass species can serve as reservoirs of the virus and a source of virus introduction into field crops. The grass host species of WSMV are shown in Table 1. In the present report, we describe five species, namely, A. capillaris, A. odoratum, B. sterilis, H. lanatus and H. mollis, as new hosts of WSMV, and several of the other species identified (L. multiflorum, B. japonicus, A. elatius and E. crus-galli) were confirmed to be hosts in earlier reports (Vacke et al. 1986; Coutts et al. 2008b). The broad host range of WSMV and WCMs among Poaceae species creates the potential threat of virus invasion in cereal crops, in particular winter wheat. Some of these grass species are important weeds of cereal crops, including E. crus-galli, E. repens, L. multiflorum, B. japonicas, and P. pratensis. These weeds may act as reservoirs for WSMV, facilitating its introduction into wheat crops. In our survey, WSMV was detected solely in winter wheat, and its incidence was recorded as approximately 16% in the tested field. However, no evidence exists for the Czech Republic regarding whether such restriction of the virus in crops occurs through seed transmission, as was reported in New Zealand (Lebas et al. 2009). Host resistance to WSMV infection might be an effective method of disease control. The sources of resistance to WSMV are temperature-sensitive (Fahim et al. 2012). Cultivars derived from Wsm2, including RonL (Seifers et al. 2007), Snowmass (Haley et al. 2011), Clara CL (Martin et al. 2014) and Oakley CL (Zhang et al. 2015a), were reported to be resistant to WSMV isolates from the USA. The cultivars Mace, Millennium and Pronghorn were shown to be cultivated to a certain level of tolerance to WSMV in Nebraska, USA (Wegulo et al. 2008). Thus far, no evidence of resistance in commonly grown wheat cultivars in Central Europe is available. Quantitative analysis of virus titre using real-time qpcr (Balaji et al. 2003) seems to be very effective for characterising the level of resistance and provides significant enhancement in addition to biological assays (Jarošová et al. 2013). Field experiments involving mechanical inoculation of WSMV showed a low level of WSMV uptake in some winter cultivars, such as Caldwell, Cubus, Florida, Matchball and Cim rana. We previously showed that virus titre (copy number) is closely correlated with a high level of cereal (wheat and barley) resistance to Barley yellow dwarf virus (Jarošová et al. 2013). The low WSMV uptake in the above-mentioned cultivars may also be associated with potentially significant resistance to the virus under field conditions (Fig. 4). However, for more thorough evaluation of the resistance of the tested cultivars, multiple traits and characteristics need to be considered. Furthermore, host resistance to WSMV in wheat has been reported to be temperature-sensitive (Seifers et al. 2013), which is a major concern for the development of new cultivars with stable resistance against virus-host interactions under field conditions. Hence, environmental conditions (i.e., temperature) during the growing session play a significant role in the stability of wheat resistance to WSMV in the field. 5. Conclusion Our results provide significant evidence that the presence of WSMV in winter wheat crops, together with Wheat dwarf virus and Barley yellow dwarf virus, may cause serious problems with respect to crop protection. Grass species, in particular the weed hosts described here, act as reservoirs of WSMV and are possibly the key source of the introduction of WSMV into winter wheat (cereal crops). Quantitative analysis of virus uptake in wheat cultivars and one cultivar of triticale showed that some cultivars, including Caldwell, Cubus, Florida, Matchball and Cim rana, had a low virus titre. Our results suggest that the low virus titre detected in these cultivars may be associated with a significant level of resistance to WSMV under field conditions. However, further analysis based on multiple resistance traits associated with yield loss should be performed to more thoroughly evaluate the resistance status of the cultivars. Acknowledgements We thank Mrs. Zuzana Červená (Division of Crop Protection and Plant Health, Crop Research Institute, Czech Republic) for her technical assistance and Dr. Fousek and Dr. Neubau-

8 *** et al. Journal of Integrative Agriculture 2017, 16(0): erová (Division of Crop Protection and Plant Health, Crop Research Institute, Czech Republic) for their help with some experiments. This work was supported by the grants from the Ministry of Agriculture, Czech Republic from projects QJ (50%) and RO0415 (50%). References Atkinson T G, Grant M N An evaluation of streak mosaic losses in winter wheat. Phytopathology, 57, Balaji B, Bucholtz D B, Anderson J M Barley yellow dwarf virus and Cereal yellow dwarf virus quantification by realtime polymerase chain reaction in resistant and susceptible plants. Phytopathology, 93, Baley G J, Talbert L E, Martin J M, Young M J, Habernicht D K, Kushnak G D, Berg J E, Lanning S P, Bruckner P L Agronomic and end-use qualities of Wheat streak mosaic virus resistant spring wheat. Crop Science, 41, Brakke M K Wheat streak mosaic virus. CMI/AAB Descriptions of Plant Viruses. No. 48. 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