The presence of Turnip yellows virus in oilseed rape (Brassica napus L.) in Serbia

Dragana Milošević1*, Maja Ignjatov1, Zorica Nikolić1, Ivana Stanković2, Aleksandra Bulajić2, Ana Marjanović-Jeromela1 and Branka Krstić2 1Institute of Field and Vegetable Crops, Maksima Gorkog 30, 21 000 Novi Sad, Serbia 2University of Belgrade, Faculty of Agriculture, Nemanjina 6, 11080 Belgrade, Serbia *Corresponding author: dragana.milosevic@nsseme.com Received: 30 December, 2015 Accepted: 30 January, 2016


The presence of Turnip yellows virus in oilseed rape (Brassica napus L.) in Serbia INTRODUCTION
Oilseed rape (Brassica napus L), also known as rapeseed or canola, is a bright yellow flowering plant, a member of the Brassicaceae family. Due to its high oil and protein contents in seed, oilseed rape is mainly grown for the production of vegetable oil for human consumption, as an animal feed, and a biodiesel. The main producers of oilseed rape are China, the EU, Canada, and India. World production of oilseed rape is growing rapidly, increasing from 36 million tones in 2004 to an estimated 58.4 million tonnes in the 2010-2011 crop season (USDA, 2011). Furthermore, oilseed rape is a very profitable crop with very high oil yields per unit area, and for that reason the production of this crop is increasing in Serbia. The harvest area of this important oil-producing crop increased in Serbia from 12,012 ha in 2011/12 to 13,000 ha in 2015/16 (Association for the Promotion of Production and Exports of Grains and Oilseeds, www.zitasrbije.rs).
More than 12 viruses from different viral genera have been reported to infect oilseed rape and cause different levels of losses in its production. However, economically the most important viruses are: Beet western yellows virus (BWYV), Cauliflower mosaic virus (CaMV), and Turnip mosaic virus (TuMV) (Latham et al., 2003;Shahraeen, 2012). There are reports of oilseed rape yields being severely affected by BWYV, CaMV and TuMV, sustaining reductions of 70-79% (Hardwick et al., 1994).
BWYV was first reported in Tasmania in the early 1980s and has been detected in a variety of crops, including legumes . Many of these hosts were asymptomatic . Some of the viruses previously described as BWYV, but shown not to infect sugarbeet (Beta vulgaris L.), have now been re-classified as a separate species in the genus Polerovirus, family Luteoviridae, under the name Turnip yellows virus (TuYV) (Graichen & Rabenstein, 1996;Hauser et al., 2000;Hauser et al., 2002). TuYV as a virus species has been ratified by the International Committee for the Taxonomy of Viruses (Mayo, 2002).
TuYV has a wide range of hosts and can infect species from at least 13 plant families, including many important plant species (cauliflower, cabbage, spinach, lettuce, etc.), but the virus is particularly significant as a pathogen of oilseed rape (Jay et al., 1999;Hill et al., 1989). The diverse range of cultivated plants and weed species susceptible to TuYV complicates its epidemiology by expanding the potential reservoirs in which the virus can overwinter and thus providing sources for future viral outbreaks (Stevens et al., 1994;Latham et al., 2003).
Oilseed rape plants infected with TuYV produce a wide range of symptoms -most of which go unnoticed because they resemble those caused by stress and nutrient-deficiency. The most common symptoms are red discoloration along the edges of infected leaves, followed by an intense chlorosis of the whole blade, which becomes hard and brittle. Infected plants remain dwarfed, and have small roots (Duffus & Russell, 1972).
Considering the importance of oilseed rape, the increasing spread of TuYV on various types of fam. Brassicae, and the common presence of many aphids that are vectors of the virus, TuYV is potentially a limiting factor for successful production of oilseed rape in Serbia. After the first detection of TuYV infected oilseed rape (Milošević et al., 2015), a survey was conducted in the main oilseed rape-growing areas of Serbia in order to survey the incidence and distribution of TuYV, and possible presence of other important oilseed rape infecting viruses, TuMV and CaMV, and to perform molecular characterization of the obtained isolates by comparing them with isolates from all over the world.

Survey and sample collection
During 2014, a total of 86 samples of oilseed rape plants showing virus-like symptoms were randomly collected in six localities in three districts of the Vojvodina Province: West Bačka (Sombor, Crvenka and Kula), Srem (Sremska Mitrovica) and North Banat (Kikinda and Zrenjanin). The samples consisted of symptomatic leaves from different parts of each plant, which were collected and placed in plastic bags, and stored at 4°C until ELISA testing (up to 5 days), or stored at -20 o C until RNA extraction.

Serological detection
The collected samples were tested for the presence of the most common oilseed rape viruses: Turnip yellows virus (TuYV), Cauliflower mosaic virus (CaMV), and Turnip mosaic virus (TuMV), using commercial doubleantibody sandwich (DAS)-ELISA diagnostic kits (Loewe Biochemica, Germany) according to the manufacturer's instructions. Plant tissues were prepared in an extraction buffer at a ratio of 1:10 (wt/vol). After incubation with p-nitrophenyl phosphate (Sigma-Aldrich, St. Louis, MO) at room temperature for 2 h in the dark, absorbance at 405 nm was measured with an ELISA microplate reader (Multiscan Ascent, Finland). The samples were considered positive if their absorbance value was twice as high as the negative control. Commercial positive and negative controls were included in each ELISA plate.

Aphid transmission
For aphid-borne TuYV inoculation, nymphs of Myzus persicae (Sulzer) were allowed to feed on the leaves of six selected serologically positive samples, i.e. one sample from each locality, over an acquisition access period (AAP) of 24 h. Groups of 5-7 aphids for each of the six isolates were then placed onto three plants of each Capsella bursa-pastoris, Physalis floridana, and B. napus cv. 'Banaćanka' for a 4-day inoculation access period (IAP). The plants with aphids were placed into separate cages under controlled conditions, at 22°C, for a 16 h photoperiod and watered regularly.

RT-PCR detection
The presence of TuY V in oilseed rape plants was further confirmed by the conventional reverse transcription (RT)-PCR. Total RNAs were extracted by the RNeasy Plant Mini Kit (Qiagen, Hilden, Germany) from 100 mg of leaf tissue of six selected samples originating from different localities. RT-PCR was performed using the One-Step RT-PCR Kit (Qiagen) with primers TuYVorf0F and TuYVorf0R amplifying the P0 gene, the most useful for delineation of Poleroviruses (Schubert et al., 1998). The RT-PCR reaction mixture included 400 μM of each of the four dNTPs, 1 μl of RT-PCR enzyme mix, 0.6 μM of each primer, and 1 μl of extracted RNA in a final volume of 25 μl. Amplifications were performed in a thermal cycler (Eppendorf, Germany) with the following cycling parameters: reverse transcription at 50°C for 30 min and an initial PCR denaturation step at 95°C for 15 min, followed by 35 cycles consisting of a denaturation step for 30 s at 94°C, primer annealing for 30 s at 55°C, and extension for 60 s at 72°C. The final extension was performed at 72°C for 10 min. The amplified products were analyzed by 1% agarose gel electrophoresis and visualized under a UV transilluminator. A tissue sample from the healthy oilseed rape leaf was used as a negative control in RT-PCR assays.

Sequencing and phylogenetic analysis
After purification with a QIAquick PCR Purification Kit (Qiagen), the amplified products from one selected isolate (119-TuYV) were sequenced directly in both directions, using the same primers as in RT-PCR. Additionally, a previously identified oilseed rape TuYV isolate 114-TYuV (Milošević et al., 2015) was also included in the investigation. The sequence of the Serbian TuYV isolate was compared with the previously reported isolates available in GenBank (http://www.ncbi.nlm.nih. gov/BLAST/), using the ClustalW program (Thompson et al., 1994) and MEGA5 software (Tamura et al., 2011). A p-distance model was applied for nucleotide (nt) and deduced amino acid (aa) sequence analyses.
A phylogenetic tree was constructed using the TuYV P0 gene sequence generated in this study, one generated in another study by Milošević et al. (2015) and 11 P0 sequences of TuYV, Beet western yellows virus, Beet chlorosis virus, and Cucurbit aphid-borne yellows virus isolates retrieved from GenBank (Table 1), using the Maximum parsimony method implemented in MEGA5. Intra-and inter-group diversity values were calculated as the average genetic distance using Kimura 2-parameter model Gamma distributed (K2+G), which was chosen as the best-fitting model of nt substitution.

Virus detection and symptomatology in the field
During our visual inspection of oilseed rape fields in 2014, similar symptoms were observed in all inspected localities with disease incidence ranging from 10 to 70%. Oilseed rape plants exhibited virus-like symptoms including the reddening of leaf margins (Figure 1a) and interveinal yellowing (Figure 1b).  (Table 2). After the first detection of TuYV in 20 tested oilseed rape samples originating from Crvenka locality, the virus was serologically detected in additional 40 oilseed rape samples collected from another five localities: Sombor, Kula, Sremska Mitrovica, Kikinda and Zrenjanin. The highest incidence of TuYV was in Crvenka (100% samples tested positive) and Sremska Mitrovica localities, where the virus was confirmed in 11 out of 15 tested samples (73.33%). In Kikinda locality, TuYV was detected in 70% of all tested samples, while the virus was confirmed in 66.66% of the tested samples in Zrenjanin locality. In Kula locality, the virus was detected in 9 out of 15 oilseed rape samples (60%), while the presence of the virus was confirmed in only 5 out of 14 tested samples (35.71%) in Sombor locality.

Host range
The virus isolates from naturally infected oilseed rape plants, one from each locality including the isolate described by Milošević et al. (2015), were successfully transmitted by M. persicae to the test plants. All inoculated C. bursa-pastoris plants exhibited leaf reddening and stunting, while all inoculated P. floridana plants showed a very mild intervenial chlorosis 5 weeks post-inoculation (wpi). The virus was successfully transmitted to B. napus cv. 'Banaćanka', which reacted with a mild yellowing symptom 6 wpi. All inoculated plants of each species tested positive for TuYV using ELISA test.

Molecular detection, identification and phylogenetic analysis
The results of serological analyses of TuYV presence in oilseed rape in Serbia was further confirmed by the molecular RT-PCR method using the specific primers TuYVorf0F and TuYVorf0R, which amplify a fragment of the TuYV P0 gene. These primers successfully detected the presence of TuYV in all tested samples and amplified cDNA fragments of predicted size. One clear band of 780 bp was visible in all oilseed rape plants assayed, while no amplification products were observed in the healthy controls.
After purification, the RT-PCR product derived from the isolate 119-TuYV was directly sequenced in both directions using the same primer pair as in RT-PCR, and deposited in GenBank (GenBank Accession No. KU351664). Sequence analysis of the P0 gene, conducted with MEGA5 software, revealed 99.7% nt identity (100% aa identity) between the two Serbian TuYV isolates from oilseed rape. One Serbian isolate (119-TuYV) showed the highest nucleotide identity of 99% (100% amino acid identity) with TuYV-GB isolates from England (AF168608).
A maximum parsimony tree (Figure 2), reconstructed using partial sequences of the P0 gene isolates, revealed that the TuYV isolate characterized in this study and the previously identified Serbian TuYV isolate (Milošević et al., 2015), as well as the selected sequences of 11 characterized TuYV, BWYV, BChV, and CABYV isolates retrieved from GenBank database clustered into four groups depending on virus species, with high bootstrap values (100%). Genetic diversity among the four molecular groups of isolates ranged from 0.516±0.038 to 0.583±0.212, while diversity within each group was: 0.068±0.008 (TuYV), 0.119±0.013 (BWYV), 0.002±0.002 (BChV), and 0.119±0.013 (CABYV). An analysis of P0 gene sequence data for a subset of these isolates showed that they clustered with known TuYV and were distinct from BWYV isolates.

DISCUSSION
Oilseed rape is now the second most important source of vegetable oil in the world (Raymer, 2002). Oilseed rape oil is of high quality, rich in proteins (over 23%) and with a suitable composition of unsaturated fatty acids and low percentage of saturated fatty acids (Ebrahim-Ghomi, 2014). Accordingly, the production of oilseed rape and especially its limiting factors, including viruses, are attracting great attention in all growing regions. Due to its importance, cultivation is increasing annually in Serbia and oilseed rape is grown on close to 10,000 ha with a production volume of over 31,000 t in 2014 (Statistical Office of the Republic of Serbia, 2014). The emergence of new and destructive pathogens, such is TuYV, is of great importance. As information about the variety of symptoms in oilseed rape, virus distribution, and above all its incidence were not available, a survey was conducted in the main oilseed rape-producing areas in the Province of Vojvodina. After the first reported outbreak of TuYV in oilseed rape in the locality of Crvenka (Milošević et al., 2015), the virus was in this study detected serologically in five other localities in 2014. The presence of TuYV was serologically detected in 69.77% of the tested samples and all samples were negative for CaMV and TuMV. The highest incidence of TuYV was found in the localities Crvenka (100%) and Sremska Mitrovica (73.33%). The incidence was lowest in Sombor locality, but still with a significant percentage of samples (35.71%). In almost all localities, a small number of samples tested negative for the presence of TuYV, and other tested viruses, CaMV and TuMV, and remained with unknown ethiology. Further research focusing on the presence of possible other viruses is ongoing. In major oilseed rape growing regions, incidence reports for TuYV have been variable and ranging from 10 to 85% (Walsh et al., 1989;Hardwick et al. 1994;Jay et al., 1999). In Australia, the presence of TuYV in oilseed rape crops has led to yield decreases of up to 46% (Jones et al., 2007), while TuYV-infected crops in Germany have been reported to yield between 12% and 34% fewer seeds than virus-free plants (Graichen & Schliephake, 1999). Most probably, the high incidence of TuYV infection in oilseed rape over a period of several years is related to its wide host range and a great number of aphid species which are able to transmit TuYV to oilseed rape plants.
In the latest host range studies, a TuYV isolate from oilseed rape was able to infect 65 out of a total of 130 species, among them many common weeds and several cultural crops (Graichen & Schliephake, 1999). Under experimental conditions, 17 of 24 tested aphid species were able to transmit the TuYV virus (Schliephake et al., 2000). Epidemiological data for TuYV in Serbia are still to be investigated and are of great importance considering the virus incidence detected in this research. Efficient spreading of TuYV by several aphid vectors, and such high incidence and distribution in Serbia, imply that TuYV could become an impediment to successful production of oilseed rape in our country. Moreover, considering that the virus is infective to other species of the family Brassicaceae, as well as numerous weeds, further research is needed to detect the span of this virus and inoculum sources in nature.
Red discolouration at the margins can be the first symptom of TuYV infection of oilseed rape, followed later by conspicuous discolouration of the whole leaf (Graichen & Peterka, 1999), and both symptoms were observed on oilseed rape plants in Serbia. The incidence of TuYV in oilseed rape crops is closely related with the activity of flying aphid vectors, while virus spreading depends on the number and movement of vectors in the crop (Walsh & Tomlinson, 1985). In Germany, a high intensity of TuYV infection was observed in oilseed rape crops during 1995-1996, followed by a high activity of aphids in the autumn of 1995.
The Serbian isolate (119-TuYV) from oilseed rape showed the highest nucleotide identity of 99% (100% amino acid identity) with a TuYV-GB isolate from England (AF168608), which is consistent with the official criterion of separation of species in the genus. According to King et al. (2011) the species demarcation criteria for the genus Polerovirus include differences in amino acid sequence identity of any gene product greater than 10 %.
TuYV has been shown to be widespread in all major crop-producing areas in Vojvodina at high incidence, and it is the only virus detected in a large number of samples. This study provides data which were missing about viruses in oilseed rape crops in Serbia. Considering that the production of oilseed rape is growing rapidly in Serbia, the occurrence of TuYV could become an impediment to successful production of that crop. As TuYV is often found in various crops (Graichen & Rabenstein, 1996;Stevens et al., 1994), and is readily transmitted in a non-persistent manner by aphids, constant monitoring of TuYV status and its presence in Serbia is necessary. Therefore, future research of epidemiology and formation of natural reservoirs of major viruses and the most efficient aphid species is of utmost importance for determining and implementing effective control measures.