Cereal Research Communications 36(2), pp. 215–224 (2008) DOI: 10.1556/CRC.36.2008.2.2

Detection of Cereal Viruses in Wheat (Triticum aestivum L.) by Serological and Molecular Methods Z. ÁY1, Z. KERÉNYI2, A. TAKÁCS3, M. PAPP1, I.M. PETRÓCZI1, R. GÁBORJÁNYI3, D. SILHAVY2, J. PAUK1* and Z. KERTÉSZ1 1

Cereal Research Non-Profit Company, Szeged, Hungary 2 Agricultural Biotechnology Center, Gödöllõ, Hungary 3 Georgikon Faculty of Agriculture, University of Pannonia, Keszthely, Hungary (Received 4 October 2007; accepted 12 February 2008)

The reliable monitoring of field virus infections of crop species is important for both farmers and plant breeders. The aim of this study was to detect virus infections of winter wheat in the 2006/2007 season. Twelve well-known winter wheat varieties were sown on two different dates (11th of October and 3rd of November 2006). Leaves of two individuals from each genotype were collected on 23rd of April 2007 to detect the virus infections (Barley stripe mosaic virus – BSMV, Barley yellow dwarf virus – BYDV-PAV, Wheat dwarf virus – WDV and Wheat streak mosaic virus – WSMV) after an extra mild autumn- and wintertime. Virus infections were detected by enzyme-linked immunosorbent assay (ELISA) and polymerase chain reaction (PCR). The aphid-transmitted BYDV-PAV was found frequently whereas other viruses were presented very rarely or were not detected. Forty-six per cent of the tested wheat plants proved to be infected by BYDV-PAV in ELISA, while using PCR, the virus infections with BYDV-PAV was found in 58% of the samples. Further, these results suggest that the optimal sowing time is critical in the control of cereal virus diseases, and additionally, that wheat varieties respond to the virus infections differently. Keywords: winter wheat, genotype, virus, ELISA, PCR

Introduction Many cereal viruses were described in the second half of the 20th century. The most important challenge was to identify and detect different pathogens. Improved identification methods were developed which were more suitable for this task. At first, the detection of the viruses was done visually, based on disease * Corresponding author; E-mail: [email protected]

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symptoms. With the advent of electron microscopic methods and their diagnostic application viruses could be separated on the basis of their composition and architecture. The visual detection of viruses was further advanced by the new serological and molecular techniques. Virus infections in cereals were detectable using the viral coat protein as antigen (Lister and Rochow 1979). The principal of the serological reactions is the recognition of foreign proteins in the blood of a homoisothermic animal, which induces the production of specific antibodies in the blood serum. Antigens are recognized by their antibodies in the immune response, and make possible the exact identification of the pathogen (Long and Kindt 1988; Horváth and Gáborjányi 1999). The most important cereal viruses were easily detectable by enzymelinked immunosorbent assay (ELISA) according to Clark 1981; Torrance 1991; Sukhacheva et al. 1996; Achon and Serrano 2006. In hybridization probes, nucleic acid samples are separated according to size then blotted onto a nylon- or nitrocellulose-based filter. Nucleic acids bound to the filter are hybridized by a probe giving colour reaction or by radioactive probes. Hybridization methods have been successfully used to detect many different viruses, with the first being the Barley yellow dwarf viruses (BYDVs) (Habili et al. 1987; Figueira et al. 1997; Liu et al. 2007). The most efficient and well-known virus diagnostic procedure was elaborated by the polymerase chain reaction (PCR) based on target amplification (Schweitzer and Kingsmore 2001). The sensitivity of the reaction depended on the tested virus and the origin of plant tissue. However, the sensitivity of the PCR based methods was higher than the above-mentioned methods (Figueira et al. 1997; Mumford et al. 2004; Ratti et al. 2004). PCR was applied successfully in the detection of cereal viruses using suitable primer pairs. For instance, all strains of BYDV (Robertson et al. 1991; Fabre et al. 2003a) and many other viral pathogens of cereals (Clover and Henry 1999; Ratti et al. 2004) were detected by PCR. An additional benefit of the PCR based methods was the possibility of simultaneous diagnosis and detection of mixed virus infections (French and Robertson 1994; Gitton et al. 1999; Malmstrom and Shu 2004; Mumford et al. 2004). Wheat is exposed to many pathogens because of its wide geographical spread. Although sixty-six viruses are able to infect grasses (Lapierre and Signoret 2004), only a few of them causes economically important yield depression on wheat. In Hungary, the four most dangerous cereal viruses are the Wheat dwarf virus (WDV), the Barley stripe mosaic virus (BSMV), the Wheat streak mosaic virus (WSMV) and the BYDVs (Gáborjányi et al. 1991; Pocsai et al. 2002). WDV is a single-stranded DNA virus while BSMV, WSMV and BYDVs are single-stranded RNA viruses. In Hungary, BYDV was first identified on winter barCereal Research Communications 36, 2008

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ley (Szirmai 1967) then on winter wheat, too (Szunics and Szunics 1980). The occurrence of BSMV was first described by Milinkó and Remete (1984), and characterized by Nagy and Gáborjányi (1991). WSMV was observed and identified by Pocsai and Barabás (1985), and confirmed by Nyitrai and Gáborjányi (1988). The presence of WDV was detected relatively late at the end of the 1980s (Gáborjányi et al. 1988). Economically less important viruses, such as Agropyron mosaic virus (AgMV) and Ryegrass mosaic virus (RyMV) also existed in cereals (Gáborjányi 1991). Recently Brome streak mosaic virus (BrSMV) a new emerging virus was described from yellow nutsedge (Takács et al. 2008). The presence of this pathogen in wheat was suspected from earlier serological studies (Mesterházy et al. 2002). In this paper, data on the above-mentioned four important cereal viruses (WDV, BSMV, BYDV-PAV and WSMV) are published. Many different wheat varieties were sown in the early and late autumn of 2006 (referred to as early- and late-sown plants, respectively) and then, virus infections were studied in the spring of 2007 using both by the traditional ELISA and by PCR-based methods. Materials and Methods Plant material Twelve registered wheat varieties (‘GK Élet’; ‘GK Garaboly’; ‘GK Kalász’; ‘GK Verecke’; ‘GK Ati’; ‘GK Tisza’; ‘GK Békés’; ‘GK Csillag’; ‘GK Petur’; ‘GK Hattyú’; ‘GK Holló’ and ‘GK Piacos’) were tested in Öthalom Experimental Station, by the Cereal Research Non-Profit Company in Szeged. Two different sowing times were used. The earlier was on 11th of October (early-sown) while the later on 3rd of November (late-sown). The weather was unusually mild in the autumn- and wintertime of the 2006/07 season. These conditions were especially favourable for the quick propagation of the vectors of viral pathogens. At the time of sample collection, a substantial part of the early-sown parcels showed severe virus symptoms (dwarf phenotype and general yellowing) while the late-sown others seemed to be healthy. Two individuals were selected from each wheat genotype and their leaves were collected on 23rd of April 2007. The collected leaf samples were divided for the detection of the virus infections by the two methods: ELISA and PCR.

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Serological tests Leaf samples collected for serological tests were stored for 1–4 days at 4 ºC in special plastic bags. The virus content was checked by DAS-ELISA using different antisera of BSMV, WDV, BYDV-PAV and WSMV from Loewe Biochemica (Clark and Adams 1977). Substrate absorbance was measured using a Labsystems Multiscan ELISA reader at 405 nm wavelength. Samples were considered negative if the absorbance value did not exceed three times those of the non-infected control. Molecular tests The leaf samples were separated along the major vein. The left and right sides were handled separately for the isolation of genomic DNA and total RNA. The leaves were cut into pieces and put into Eppendorf-tubes. The tubes were placed immediately into liquid nitrogen and then stored in an ultra freezer at –80 ºC. The frozen tissues were rubbed in the Eppendorf tubes with special, chilled glass staff. For the first time in Hungary, the Promega “MaxwellTM 16 Instrument” semi-automatic DNA-preparation equipment and the “MaxwellTM 16 DNA Purification Kit” (Kephart et al. 2006) were used for the isolation of genomic DNA. For the isolation of total RNA, the Promega “SV Total RNA Isolation System” kit was applied (Otto et al. 1998; Kobs 1998). Instead of the vacuum technique, the centrifuge-protocol was chosen which contained the DNase treatment, too. The concentrations of the genomic DNA and the total RNA samples were determined by spectrophotometer, and then they were set to the same concentration by nuclease free water. For the detection of the virus infections, the same concentrated nucleic acids were used as template in PCR. Each sample was PCR tested with the “Ubi 306 + Ubi 664” primer pair designed for the gene ubiquitin-ligase. These reactions provided information about the PCR-quality of the samples. Traditional PCR was assembled for the detection of the DNA virus (WDV) while the RNA viruses (BSMV, BYDV-PAV and WSMV) were detected by one-step reverse transcriptase (RT)-PCR. The solutions of “Fermentas” were used for the traditional PCR and the “Qiagen One Step RT-PCR Kit” for the RT-PCR. RT-PCR mixtures were supplemented with “RiboLockTM Ribonuclease Inhibitor”. Plasmid DNAs and nucleic acids isolated from artificially infected plants served as positive controls for the PCR and RT-PCR reactions. The “BioRad iCyclerTM Thermal Cycler” equipment was used for the reactions. Primers were so developed for the RT-PCR that the simultaneous detection of the mixed virus infections (BSMV+BYDV– PAV+WSMV) was possible with the same program. Primers were designed to Cereal Research Communications 36, 2008

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cover the most conserved regions of each virus, thus it is likely that our primer pairs can detect all strain of a virus. Eight µl PCR products and two µl brome-phenol-blue paints were mixed. The mixture was taken on to agarose gel prepared from 0.5× TBE buffer. The concentration of the gels varied between 0.9–1.2% depending on the length of the fragments. In each case, 90 Voltages were applied during electrophoresis. Results Serological tests One part of the divided leaf samples was examined by DAS-ELISA. From the late-sown plant material, only one variety (‘GK Élet’) showed detectable infection with BYDV-PAV while the other three viruses were undetectable. Contrast with this, the early-sown wheat varieties were heavily infected. BYDV-PAV was detected in 46% of the tested plants. Both individuals contained the virus in the following three varieties: ‘GK Garaboly’; ‘GK Kalász’ and ‘GK Ati’ while in the case of ‘GK Élet’; ‘GK Verecke’; ‘GK Békés’; ‘GK Holló’ and ‘GK Piacos’ only one of the two samples showed infection with BYDV-PAV. Four varieties: ‘GK Tisza’; ‘GK Csillag’; ‘GK Petur’ and ‘GK Hattyú’ proved to be uninfected by BYDV-PAV (Table 1). Other three viruses were detected at much lower frequencies even in the early-sown parcels. WSMV infection was found in ‘GK Holló’ and ‘GK Piacos’ whereas WDV infection was detected only in one sample of ‘GK Piacos’. BSMV was not detected at all. Two wheat varieties: ‘GK Holló’ and ‘GK Piacos’ showed mixed virus infections. BYDV-PAV occurred together with WSMV in the former variety, while in the latter, WDV, BYDV-PAV and WSMV were present also in mixed infection. Molecular tests Half part of the leaf samples were examined by PCR assays. High quality nucleic acids were purified from all wheat samples. Before virus specific PCR, nucleic acid samples were tested by the “Ubi 306 + Ubi 664” primer pair. Each DNA and RNA samples proved to be suitable for PCR. Control experiments confirmed that our primer pairs can be used efficiently to detect all the four viruses from infected wheat plants. From the late-sown parcels, only the same ‘GK Élet’ sample showed detectable virus infection which was also positive for BYDV-PAV in ELISA. According to the results of PCR, 58% of the tested plants proved to be infected by Cereal Research Communications 36, 2008

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ÁY et al.: Wheat Virus Detection Table 1. Infections with BYDV-PAV in the tested plants sown in different times Sown on 11th of October 2006 ELISA

sample 1 – + + – + – – – – – + –

Sown on 3rd of November 2006

PCR

ELISA

Wheat varieties

PCR

sample 2

sample 1

sample 2

sample 1

sample 2

sample 1

sample 2

+ + + + + – + – – – – +

– + + – + – + – – – + +

+ + + + + – + – – – + +

– – – – – – – – – – – –

+ – – – – – – – – – – –

– – – – – – – – – – – –

+ – – – – – – – – – – –

GK Élet GK Garaboly GK Kalász GK Verecke GK Ati GK Tisza GK Békés GK Csillag GK Petur GK Hattyú GK Holló GK Piacos

– negative sample + positive sample

BYDV-PAV in the early-sown plant material (Fig. 1). Both of the two individuals contained the virus in case of the following six wheat varieties: ‘GK Garaboly’; ‘GK Kalász’; ‘GK Ati’; ‘GK Békés’; ‘GK Holló’ and ‘GK Piacos’ while only one sample showed infection with BYDV-PAV in the case of ‘GK Élet’ and ‘GK Verecke’. The same four varieties: ‘GK Tisza’; ‘GK Csillag’; ‘GK Petur’ and ‘GK Hattyú’ which were negative in ELISA proved to be uninfected in PCR, too (Table 1). Moreover, in the early-sown parcels, WSMV infection was found only in one variety (‘GK Ati’). BSMV and WDV were undetectable in each case. BYDV-PAV occurred together with WSMV only in ‘GK Ati’.

1.

2.

3.

4.

5.

A

6.

7.

8.

9.

aip

dw

p

B

Figure 1. Detection of infections with BYDV-PAV by RT-PCR in early-sown (A) and late-sown (B) wheat plants. 1 and 6: GK Ati; 2 and 7: GK Tisza; 3 and 8: GK Békés; 4 and 9: GK Holló; 5: GK Piacos; aip: artificially infected plant; dw: distilled water; p: plasmid-DNA

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Discussion The virus content of the twelve wheat varieties collected from field experiment was checked by serological and molecular methods. The results of the two assays correlate well supporting that the PCR-based method can be used to detect the economically important BYDV-PAV pathogen of wheat. PCR data confirmed the results of the ELISA tests in harmony with previously published results on wheat, barley and oat (Robertson et al. 1991; Figueira et al. 1997; Mumford et al. 2004). The PCR-based method provides simultaneous detection of RNA-viruses (Gitton et al. 1999; Balaji et al. 2003; Malmstrom and Shu 2004). Regarding BYDV-PAV detection, PCR gave positive results in three samples (‘GK Békés’; ‘GK Holló’ and ‘GK Piacos’) which were negative in ELISA (Table 1). These results suggest that BYDV-PAV detection based on PCR assay was more sensitive than the corresponding ELISA method. At the detection of WDV and WSMV, three positive ELISA results were not confirmed by PCR data. Although control experiments showed that WDV and WSMV PCR assays were very sensitive, it is well known that a virus spreading within a plant is never homogeneous. It is possible that the leaf samples, which were examined by PCR, did not contain these viruses. Comparative ELISA and PCR assay should be carried out on samples containing the more frequent WDV, WSMV and BSMV viruses to demonstrate that PCR based methods are as effective as ELISA tests. In the experimental fields of Szeged over a five-year period (1994–1999), the most dangerous viral diseases were subsequently tested by the ELISA method, and BYDVs was found less frequently in comparison to BSMV, WDV and WSMV. On the other hand, mixed virus infections were detected in many individual plants (Mesterházy et al. 2002). Whereas, BYDV-PAV was the dominant single viral pathogen of wheat in the last season. BYDVs have been transmitted by aphid vectors. The extra mild autumn allowed the Rhopalosiphum and other aphid vector species to feed on winter wheat until late October. Over-propagation of the vectors led to severe infections with BYDV-PAV. The occurrence of the WDV and the WSMV was insignificant because their vectors Psammotettix alienus and Aceria species were found very rarely before 23rd of April, at the time of sample collection. The mechanically spreading BSMV was detectable by neither ELISA nor PCR methods. Differences were found among the tested wheat varieties sown in October. All of the four tests done for BYDV-PAV gave positive result in the genotypes ‘GK Garaboly’; ‘GK Kalász’ and ‘GK Ati’ while there were three positive results in case of ‘GK Békés’; ‘GK Holló’ and ‘GK Piacos’. These varieties seemed to be susceptible to BYDV-PAV. In the ‘GK Ati’; ‘GK Holló’ and ‘GK Piacos’, WDV and/or WSMV infections were found, too. Two of the four BYDV tests gave posiCereal Research Communications 36, 2008

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tive data in ‘GK Élet’ and ‘GK Verecke’ varieties. Four varieties: ‘GK Tisza’; ‘GK Csillag’; ‘GK Petur’ and ‘GK Hattyú’ proved to have a high resistance degree not only to BYDV-PAV but to all viruses in this experiment. In parallel experiments (data are not shown) two from these varieties – the ‘GK Tisza’ and the ‘GK Hattyú’ – showed typical virus symptoms. Concerning the variety resistance the present experiment is not enough to evaluate the resistance relations of different genotypes but further experiments are needed. This study concentrates only on the methodological details. The present study demonstrates that the sowing time considerably influences the spread and outbreak of cereal viruses. WDV and WSMV were found exclusively in the early-sown samples and in these samples, BYDV-PAV occurred significantly more often than in the late-sown ones. These results support that the early sowing time involves the risk of a mixed virus infection in autumn (Cisar et al. 1982; Perry et al. 2000). In springtime, these infected plants will be underdeveloped and this phenomenon can lead to yield depression (Jensen and D’Arcy 1995). A precise technology during wheat production can help to protect plants and reduce damages (Fabre et al. 2003b; Pribék et al. 2005). Finally, it can be concluded that the results obtained by the two methods are well correlated. It was found that while the late-sown wheat population was practically virus-free and the early-sown was frequently infected. Our data show that in the spring of 2007, the BYDV-PAV was the predominant wheat virus in South-Hungary. Acknowledgements Authors express their thanks for subsidy of “Búzakalász Konzorcium” (KPI 4 064 04) and NAP_BIO2006ALAP3-01435/2006 coordinated by Prof. D. Dudits. References Achon, M.A., Serrano, L. 2006. First detection of wheat dwarf virus in barley in Spain associated with an outbreak of barley yellow dwarf. Plant Disease 90:970. Balaji, B., Bucholtz, D.B., Anderson, J.M. 2003. Barley yellow dwarf virus and cereal yellow dwarf virus quantification by real-time polymerase chain reaction in resistant and susceptible plants. Phytopathology 93:1386–1392. Cisar, G., Brown, C.M., Jedlinski, H. 1982. Effect of fall or spring infection and sources of tolerance of barley yellow dwarf of winter wheat. Crop Sci. 22:474–478. Clark, M.F. 1981. Immunosorbent assays in plant pathology. Annual Review of Phytopathology 19:83–106.

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