Three digoxigenin-labeled cDNA probes for specific detection of the natural population of Barley yellow dwarf viruses in China by dot-blot hybridization

Journal of Virological Methods 145 (2007) 22–29

Three digoxigenin-labeled cDNA probes for specific detection of the natural population of Barley yellow dwarf viruses in China by dot-blot hybridization Yan Liu, Bo Sun, Xifeng Wang ∗ , Chuanlin Zheng, Guanghe Zhou State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, No. 2, West Yuan Ming Yuan Road, Beijing 100094, PR China Received 6 December 2006; received in revised form 30 April 2007; accepted 1 May 2007 Available online 11 June 2007

Abstract Three digoxigenin-labeled cDNA probes complementary to the coat protein (CP) and read-through protein gene sequences of Barley yellow dwarf virus – one each for three species, namely BYDV-GAV, GPV, and PAV – were synthesized for developing a specific and sensitive dot-blot hybridization detection assay for total RNA extracts from field-infected wheat plants. The sensitivity limit for BYDV-GAV, GPV, and PAV probes corresponded to 25 ␮g, 31.25 ␮g, and 62.5 ␮g tissue/spot, respectively. The frequencies for each of the three species determined that BYDV-GAV was the most prevalent in 269 wheat samples collected from 5 agro-ecological areas in China during 2004–2006. The high sensitivity and reliability of the molecular hybridization assay described introduce an important alternative to serological methods for detecting BYDV. This is especially important in less developed countries like China, where appropriate antibodies for BYDV are not available. © 2007 Elsevier B.V. All rights reserved. Keywords: Barley yellow dwarf viruses (BYDVs); Primer; RT-PCR; DIG-labeled cDNA probe; Dot-blot hybridization assay

1. Introduction Barley yellow dwarf viruses (BYDVs), which are transmitted naturally by at least 25 species of aphids in a highly specific, circulative, and non-propagative manner, infect a wide range of species of Gramineae, including barley, wheat, oats, and many wild and cultivated grasses (Gray and Gildow, 2003). Early work by Rochow and others distinguished five different strains of the virus by their primary aphid vector (Rochow, 1969; Rochow and Muller, 1971); these are currently assigned to family Luteoviridae, which contains three genera, namely Luteovirus, Polerovirus, and Enamovirus (D’Arcy and Domier, 2005). BYDVs comprise BYDV-PAV, BYDV-MAV, and BYDV-PAS in the genus Luteovirus; Cereal yellow dwarf virus-RPV (CYDVRPV, formerly BYDV-RPV) and CYDV-RPS in Polerovirus, and BYDV-SGV, BYDV-GPV, and BYDV-RMV, which are yet to be assigned to any particular genus.

∗

Corresponding author. Tel.: +86 10 62815928; fax: +86 10 62896114. E-mail address: [email protected] (X. Wang).

0166-0934/$ – see front matter © 2007 Elsevier B.V. All rights reserved. doi:10.1016/j.jviromet.2007.05.006

Symptoms of infection by BYDVs consist typically of yellowing or reddening of leaves and stunted growth, easily confused with those caused by other biotic and abiotic agents. For this reason, and because several species of the virus occur together in the field, detection and diagnosis of BYDVs is an important area of study, which has so far included biological, biochemical, and biophysical techniques (Rochow, 1969; for a review, see Smith and Barker, 1999); serological assays (for a review, see French, 1995); and nucleic acid techniques (Miller et al., 1988; Chalhoub et al., 1994; Rizzo and Gray, 1990; Canning et al., 1996; Chang et al., 1999). The early techniques were based on biological properties of the viruses, mainly their transmission by aphid vectors. Rochow (1969) was the first to group BYDVs from New York into strains based on efficiency of transmission and specificity, and aphid transmission later formed the basis for identification of many luteoviruses. Variations in virus–aphid interactions were reported by others (Guo et al., 1997; Moon et al., 2000). Now, there is a wide variety of techniques available for the detection and diagnosis of luteoviruses, including biological techniques, different types of ELISA, dot-blot hybridization, RT-PCR, etc. Comparative studies indicated that RT-PCR, although the most sensitive

Y. Liu et al. / Journal of Virological Methods 145 (2007) 22–29

of these methods, was expensive, time-consuming, and prone to give conflicting results because of contamination whereas ELISA techniques, although more economical and better suited for work with large numbers of samples, were limited by the supply and quality of antisera and specific monoclonal antibodies, as well as by the type of sampling (for a review, see French, 1995). The use of nucleic acid probes for the detection of BYDVs was first reported in 1986 by Waterhouse et al., who used 32 Plabeled probes prepared from cDNA clones of BYDV-PAV and CYDV-RPV to detect purified virus RNA and virus RNA from infected plants. The 32 P-labeled probes were about 10 times more sensitive than standard ELISA for detection of BYDV-PAV in infected plants. The principal advantage of this assay is that it does not need any virus-specific antibodies, which are difficult to produce and in short supply, while the major disadvantages are the relative difficulty in preparing nucleic acid samples and the use of radioactive compounds. In the latter, non-radioactive probes can be prepared in much the same way as the 32 P-labeled probes, except that the nucleotides used to synthesize the probes were labeled with either biotin (Habili et al., 1987; Fouly et al., 1992) or digoxigenin (Lemaire et al., 1995). Wheat is the major crop infected by BYDVs. The Chinese isolates of BYDVs were divided into four species in Rochow’s system, namely GAV, PAV, GPV, and RMV (Zhou et al., 1984, 1986). BYDV-GAV, transmitted most efficiently by both Schizaphis graminum and Sitobion (formerly Macrosiphum) avenae, is serologically related with BYDV-MAV. BYDV-GPV, transmitted most efficiently by both S. graminum and Rhopalosiphum padi, did not serologically cross-react with the antibodies of five BYDV species from New York. Although PAV-CN is serologically related with BYDV- PAV, its coat protein gene shared only ∼71–74% identities with other isolates of BYDV-PAV (Jia et al., 2003; Liu et al., 2007). BYDV-RMV was found only once in Guiyang, in Guizhou province, in 1984 by biological assay (Zhou et al., 1987). Because of low concentrations of BYDVs particles in tissues and difficulties in purifying the virus, specific antibodies against Chinese species of BYDVs have not been obtained so far. The detection and diagnosis of BYDVs in the laboratory and in the field have been limited by the lack of specific antibodies and efficient diagnostic techniques for use in field assays. The objective of this work was to develop pairs of specific primers based on the sequence difference of the coat protein (CP) and read-through protein (RTP) genes. Subsequently, sensitive dot-blot hybridization tests using in vitro-transcribed DIG-labelled cDNA probes were built for specific detection of BYDV-GAV, PAV and GPV, the three BYDV species mainly found in China, and then testing them in the field as an alternative diagnostic tool for large-scale surveys as well as for long-term epidemiological and ecological studies. 2. Materials and methods 2.1. BYDV strains and plant material Laboratory isolates of BYDV-GAV, GPV, and PAV were collected in the wheat fields in Beijing during 1980s, which have

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Fig. 1. Part of the genomic map of BYDVs showing location of target sequences for primers and approximate size of fragments produced by RT-PCR.

been characterized by aphid transmission test and ELISA (Zhou et al., 1986, 1987). Leaf tissues infected with each virus isolate are kept at −20 ◦ C for nearly 20 years in our laboratory. Isolates for the experiment were regenerated from extracts of the leaf tissues firstly by allowing their respective aphid vectors to feed on them through a membrane in the dark for 48 h at 15 ◦ C and then transferring 5–10 viruliferous aphids onto healthy oat (Avena sativa cv. Coast Black) plants at the 2-leaf stage for 72 h (Rochow, 1960). The aphids were subsequently killed with a pesticide, and the inoculated oats were kept at 18 ◦ C under 20,000 lux for symptom expression. In addition, 269 wheat samples expressing BYDV-like symptoms were collected from different agro-ecological areas in China, including Northwestern area (Shaanxi, Gansu, and Ningxia provinces), Northern area (Shanxi and Hebei provinces), Central area (Henan, Shandong, and Hubei provinces), South-western area (Yunan and Guizhou provinces), North-eastern area (Liaoning province), during field surveys in the growing seasons from 2004 to 2006. These areas represented the main wheat-growing regions from which epidemics of BYDV had been reported. Whole-plant samples of wheat and oat were put into plastic bags and their roots kept moist during transport to the laboratory, namely the Plant Virology laboratory of the Institute of Plant Protection, Chinese Academy of Agricultural Sciences (CAAS, Beijing, China). Leaf samples in the form of 5-cm pieces were cut from the plants and stored in a freezer at −20 ◦ C until needed. Healthy, virus-free oat plants served as controls. 2.2. Primer design Laboratory isolates of BYDV-GPV, GAV, and PAV were selected for the preparation of the probes. Based on published coat protein (CP) and read-through protein (RTP) genes of these three BYDV species (Cheng et al., 1996; Wang et al., 2001; Liu et al., 2007), three pairs of primers, one for each species, were designed by Vector-NTI for specific amplification (Fig. 1 and Table 1). Table 1 Primers and their sequences examined in this work Primer

Sequence

GPV-F GPV-R GAV-F GAV-R PAV-F PAV-R

5 5 5 5 5 5

ATG AGT ACG GTC GCC CTT AGA A 3 TTC GTC AAG CGT AAC TGT 3 ATG AAT TCA GTA GGC CGT AGA A 3 GTC TCG GTT TCC TCC AAT GTG 3 GTA CAA GGC AAA TGG CAC GAC 3 GTT CTG CCT GTT TCC CAG CAT 3

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Y. Liu et al. / Journal of Virological Methods 145 (2007) 22–29

2.3. RNA extraction Total RNA was extracted from freshly infected leaves 2 weeks after inoculation by using the Trizol reagent according to the manufacturer’s instructions (Invitrogen, Carlsbad, CA, USA). One hundred milligrams of leaf tissue was homogenized for 2 min with 1 ml of Trizol reagent in a 2-ml RNase- and DNase-free microfuge tube. The RNA was resuspended in 40 ␮l of RNase- and DNase-free water, and its concentration determined by spectrophotometric analysis (DU Series 500UV–Vis, Beckman Coulter Inc., Fullerton, CA, USA). 2.4. RT-PCR amplification of viral RNA For reverse transcription (RT) reaction, 0.4 ␮g of plant total RNA and 50 pmol downstream primers were mixed in a microfuge tube, incubated at 90 ◦ C for 2 min, and then kept on ice for 2 min. The cDNA synthesis reaction was carried out in a total volume of 50 ␮l using 50 U RNase Inhibitor, 1 mM dNTPs, 5 mM MgCl2 , and 200 U MMLV reverse transcriptase (Promega, WI, USA) according to the manufacturer’s instructions. The PCR reaction involved 2 ␮l of the RT product, 5 pmol upstream primer, 5 pmol downstream primer, 4 nmol dNTPs, 1 U rTaq, and 2 ␮l 10 × PCR buffer (TaKaRa, Dalian, China) in a total volume of 20 ␮l and was carried out using a PCR system (BIO-RAD PTC-100, CA, USA) comprising initial incubation at 94 ◦ C for 5 min; 35 cycles of 94 ◦ C for 30 s, annealing for 45 s, and 72 ◦ C for 1 min; and final incubation at 72 ◦ C for 7 min. The annealing temperatures for the three pairs of primers, namely GAV-R/GAV-F, GPV-R/GPV-F, and PAV-R/PAV-F, were 50 ◦ C, 55 ◦ C, and 52 ◦ C, respectively. 2.5. Cloning and sequencing of cDNA fragments for each BYDV Successful amplification of fragments of the expected size was confirmed by electrophoresis in 1% (w/v) agarose gels. The fragments were then purified using a DNA gel extraction kit (TaKaRa, Dalian, China) and cloned into the pGEM-T vector (Promega, WI, USA) following the manufacturer’s protocols. The ligated vectors were transformed into an Escherichia coli strain JM110, and plasmid DNA was isolated by alkaline lysis from cultures incubated overnight. The cloned fragments were sequenced using the dideoxynucleotide chain termination method using an automated sequencer (Perkin-Elmer Applied Biosystems, CA, USA). 2.6. Labeling of species-specific probes DIG-labeled cDNA probes were generated by using the PCR DIG Probe Synthesis Kit (Roche Molecular Biochemicals, Germany) and species-specific probes for each BYDV by PCR amplification of the corresponding viral cDNA clone. The method was the same as described before except that 200 ␮M

dNTPs was substituted with 1× PCR DIG labeling mix. The presence of DIG in DNA makes the labeled probe run slower in the gel than unlabeled DNA. After gel electrophoresis, the concentration of each probe was measured by using an image analysis software, namely Kodak Digital ScienceTM 1D (Eastman Kodak Company, NY, USA). 2.7. Dot-blot hybridization assay Total RNA was extracted from the sample tissue (100 mg) by using Trizol reagent (Invitrogen, Carlsbad, CA, USA) in accordance with the manufacturer’s instructions. After precipitation with ethanol, total RNA was solubilized in 40 ␮l of DEPC-treated water. Samples of RNA (1 ␮l) were separately spotted onto nylon membranes (Hybond-N+, Amersham, Buckinghamshire, UK) soaked beforehand in 2× standard saline citrate (SSC). The nucleic acids were fixed to the membrane by baking at 120 ◦ C for 30 min. The membranes could be prehybridized immediately or used later by storing them at 2–8 ◦ C. To determine the specificity of the method, purified samples of total RNA from fresh leaves infected by the laboratory isolates of BYDV-GPV, GAV, and PAV-CN were separately applied to the corresponding membranes. Leaf extract of healthy oats served as the blank control. The extract and purified samples of RNA from BYDV-GPV, GAV, and PAV-CN were serially diluted to 1:1, 1:10, 1:20, 1:40, 1:80, 1:100, 1:200, 1:400, and 1:800 in RNA dilution buffer (H2 O:20× SSC:formaldehyde, 5:3:2) and applied to the membrane to determine the sensitivity limit of the method. The first spot of each dilution was that of total RNA purified from 2.5 mg of fresh tissue. To detect the distribution and mixed infections of each BYDV species in wheat in China during 2004–2006, total RNA from the collected field samples was extracted and spotted onto the membrane separately, the spot of each dilution corresponding to that of total RNA from 2.5 mg of the original tissue. Samples of total RNA (equivalent to 2.5 mg of fresh tissue) from the laboratory isolates of BYDV-GAV, PAV, and GPV as well as from healthy tissues were spotted onto the same membrane simultaneously. The probes were denatured at 100 ◦ C for 10 min and then chilled on ice for 5 min. Pre-hybridization and hybridization of dot-blots were performed following the manufacturer’s instructions for nucleic acid blots with PCR DIG-labeled probes (Roche Applied Science, Mannheim, Germany). The hybridization solution contained 50 ng of probes for every milliliter of solution. Hybridization was carried out overnight at 50 ◦ C. The membranes were washed twice (for 10 min each time) in 2× SSC and 0.1× SDS at room temperature and twice (for 15 min each time) in 0.5× SSC and 0.1× SDS at 68 ◦ C. The hybridized probes were immunodetected with anti-digoxigenin-AP fab fragments and then made visible for colorimetric detection with 5-bromo-4chloro-3-indolyl phosphate/nitro blue tetrazolium (BCIP/NBT), as recommended by the manufacturer (Roche, Mannheim, Germany).

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3. Results 3.1. Specificity of the three pairs of primers To evaluate the specificity of the three pairs of primers, laboratory isolates of BYDV-GPV, GAV, PAV-CN, and Wheat yellow mosaic virus (genus Potyvirus, as an out-group control) were used in the RT-PCR tests. Total RNAs were extracted from each virus-infected oat or wheat plants and analyzed by RT-PCR with three pairs of primers, namely GPV-R/GPV-F, GAV-R/GAV-F, and PAV-R/PAV-F. These primers corresponded to the conserved sequences located in the CP and RTP regions (Fig. 1), and were expected to react specifically with BYDV-GPV, GAV, or PAVCN to yield a 0.9–1.5 kb cDNA product. Accordingly, the pairs of primers GPV-R/GPV-F, GAV-R/GAV-F, and PAV-R/PAV-F generated only specific 1.3-kb, 0.96-kb and 1.47-kb RT-PCR products, respectively, corresponding to each of total RNAs from wheat plants infected by BYDV-GPV, GAV, or PAV-CN, respectively (Fig. 2, lanes 8, 2 and 14), they did not amplify any specific product from other two BYDVs, WYMV and healthy control. These results indicated that the three pairs of primers had adequate specificity because they could not amplify any cDNA product other than that for which they had been developed. 3.2. Specificity and sensitivity of dot-blot hybridization The three species of BYDV were initially detected by dotblot hybridization using the three DIG-labeled cDNA probes developed specifically for each. Each of the three sheets of nylon membranes was spotted simultaneously with purified total RNA from oat plants infected with one of the three BYDVs and that from healthy oat plants. That the two RNAs could hybridize demonstrates that the target prepared from a plant infected with a particular virus could react only with its matching probe, with very strong signals. No hybridization signals were seen between a probe and total RNA from a leaf infected by a virus that did not correspond to the probe or that from a healthy oat leaf (Fig. 3). Hybridization on the three sheets of nylon membranes soaked in serial dilutions of total RNA (equivalent to the RNA purified from 2500 ␮g, 250 ␮g, 125 ␮g, 62.5 ␮g, 31.25 ␮g, 25 ␮g,

Fig. 2. Agarose gel analysis of RT-PCR assays showing the specificity of three pairs of primers. Lanes 1 and 17, DNA size marker (DL2000, TaKaRa, Dalian, China); lanes 2–6: products of GAV, GPV, PAV, WYMV, and healthy control amplified using primers GAV-R/GAV-F; lanes 7–11: products of GAV, GPV, PAV, WYMV, and healthy control amplified using primers GPV-R/GPV-F; lanes 12–16: products of GAV, GPV, PAV, WYMV, and healthy control amplified using primers PAV-R/PAV-F.

Fig. 3. Dot-blot hybridization of purified total RNA of each BYDV-infected and healthy oat plants with three DIG-labelled cDNA probes corresponding to BYDV-GAV (line 1), GPV (line 2), and PAV (line 3). Each spot is equivalent to the total RNA purified from 2.5 mg of fresh tissue.

12.5 ␮g, 6.25 ␮g, and 3.125 ␮g fresh tissue) obtained from oat leaves infected with BYDV-GAV, GPV, or PAV, respectively, with three DIG-labeled cDNA probes corresponding to each BYDV showed a different sensitivity to the three DIG-labeled cDNA probes. The last positive and clearly detectable signal corresponded to 25 ␮g tissue/spot for GAV probe (Fig. 4, line 1), 31.25 ␮g tissue/spot for GPV probe (Fig. 4, line 2), and 62.5 ␮g tissue/spot for PAV probe (Fig. 4, line 3). No hybridization was seen with total RNA extracted from healthy oat leaves. 3.3. Field detection in wheat samples by dot-blot hybridization The three DIG-labeled cDNA probes were then used for identifying the species and their frequencies from 269 wheat samples collected from 5 agro-ecological areas of China during 2004–2006. Total RNA extracts of all samples from each year were spotted onto the three sheets of nylon membrane and hybridized with the corresponding cDNA probes, respectively. The same samples were simultaneously subjected to RT-PCR using the three pairs of primers, namely GPV-R/GPV-F, GAVR/GAV-F, and PAV-R/PAV-F. Each of the 269 samples fell into one of the five groups (Table 2). The hybridization pattern of 80 field samples collected in 2005 is shown in Fig. 5. The results for the samples collected in 2004 and 2006 are not shown because the hybridization pattern in samples collected in these years was the same as that for samples collected in 2005. Of the total, 146 samples (54.28%) reacted positively only with the GAV probe; 58 (21.56%), only with PAV probe; and 21 (7.81%), only with GPV-probe whereas 48 samples reacted negatively with all the three probes. These negative results were especially striking because they occurred on membranes where all other samples had reacted positively, which points either to B/CYBV species so far unknown in China or to other pathogens that produce similar yellowing symptoms. The mixed infections were identified by the dot-blot hybridization technique. If spots at the same location on different membranes reacted simultaneously with two or more probes, the sample was considered to be infected by two or more

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Y. Liu et al. / Journal of Virological Methods 145 (2007) 22–29

Fig. 4. Dot-blot hybridization of three DIG-labelled cDNA probes: line 1, Probe-GAV; line 2, Probe-GPV; and line 3, Probe-PAV with total RNA from different oat leaves infected, respectively, by BYDV-GAV, GPV, and PAV. Spots are equivalent to the total RNA purified from 2500 ␮g, 250 ␮g, 125 ␮g, 62.5 ␮g, 31.25 ␮g, 25 ␮g, 12.5 ␮g, 6.25 ␮g, and 3.125 ␮g of fresh tissue. Lane H indicates the total RNA from healthy tissues.

Table 2 Detection of BYDVs from field samples in China during 2004–2006 by dot-blot hybridization Year

No. of samples

BYDV-GAV (%)

BYDV-PAV (%)

BYDV-GPV (%)

Mixed infections (%)

Othersa (%)

Positive number (%)

2004 2005 2006

101 80 88

69.31 40.00 50.00

7.92 27.50 31.82

11.88 8.75 2.27

2.97 1.25 0

13.86 25.00 15.91

89.11 76.25 84.09

Total

269

54.28

21.56

7.81

1.49

17.84

83.64

a Others

mean the percentage of samples which did not react with any of three probes.

Fig. 5. Dot-blot hybridization of 80 field samples of wheat collected from different areas of China in 2005 with digoxigenin-labeled cDNA Probe-GAV (a), ProbeGPV (b), Probe-PAV (c) and sample order (d). For each sample, 1 ␮l of total RNA, equivalent to 2.5 mg of fresh tissue, was spotted. Positive controls were total RNA from oat leaves infected by the corresponding species of the virus (BYDV-GAV, GPV, and PAV) and negative controls were total RNA from healthy oat leaves.

Y. Liu et al. / Journal of Virological Methods 145 (2007) 22–29

viruses. The results showed sample 04YC14, collected in 2004 from Yuncheng in Shanxi province; 04FX-6, collected in 2004 from Fengxiang in Shaanxi province; and 05WN8, collected in 2005 from Weinan in Shaanxi province to be carrying a mixed infection of BYDV-GAV and BYDV-GPV and sample 04FX-4, collected in 2004 from Fengxiang in Shaanxi province, with the mixed infection of BYDV-GAV and BYDV-PAV. These were also considered as positive samples for each BYDV. 3.4. Incidence rates and distribution of three BYDV species Data on incidence of BYDV-GAV, GPV, and PAV in samples of wheat and oat during 2004 and 2006 are presented in Table 2. The three species occurred in varying degrees during the 3 years: the incidence of BYDV-GAV was the highest (69.31%) in 2004 and remained relatively high thereafter (40% in 2005 and 50% in 2006); that of BYDV-PAV rose from approximately 8% in 2004 to nearly 32% in 2006; and that of BYDV-GPV declined from about 12% in 2004 to approximately 2.3% in 2006. Although BYDV-GAV was the most common species of BYDV in China, BYDV-PAV appears to be on the increase in the main wheatproducing regions. When the data for all 269 samples were summarized by region (Fig. 6), the different patterns of distribution of each species in different agro-ecological areas became apparent. In Northwestern provinces (Area 1), which includes Shaanxi, Gansu, and Ningxia, the three species were found among 79 samples, BYDV-GAV being the most common (44.3%) whereas BYDVPAV (24.05%) was found mainly in Gansu province. These provinces are characterized by higher elevations and dry farmlands, at which the predominant species of aphids are usually S. graminum and S. avenae; both can transmit BYDV-GAV efficiently. In Northern provinces (Area 2), which include Shanxi and Hebei, the frequency of BYDV-GAV was very high (75.0%) but BYDV-PAV was found only in one sample. It is interesting that most of BYDV-GPV isolates (in 12 samples) were found in samples from Yuncheng, Shanxi province, an area where wheat yellow dwarf is usually very severe and S. granminum the predominant vector. In Central provinces (Area 3), which include Henan, Shandong, and Hubei, although the three species were found among 67 samples, BYDV-PAV (43.28%) was the most common species, followed by BYDV-GAV (34.33%) whereas BYDV-GPV was found only in two samples. In these lowerlying areas and irrigated lands, the predominant aphid species are

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often R. padi and S. avenae, which would be expected to transmit BYDV-PAV efficiently. In South-western provinces (Area 4), which include Yunan and Guizhou, BYDV-GAV was the most common species (58.97%) but BYDV-GPV was absent altogether. In North-eastern (Liaoning province, area 5), all 8 wheat samples collected in 2006 were those of BYDV-GAV. In the last two areas, the predominant aphid vector was often S. avenae, the aphid species that transmits such virus species. To determine the reliability of the method, all field samples were tested with RT-PCR with the corresponding specific primers, namely GPV-R/GPV-F, GAV-R/GAV-F, and PAVR/PAV-F. The numbers of positive samples were consistent with the results obtained by dot-blot hybridization assay (data not shown). 4. Discussion The present study described a non-radiocative dot-blot hybridization procedure to detect BYDV-GAV, GPV and PAV, three dominant viruses in wheat fields in China. Because the primer is a key factor for the specificity of cDNA probes, the specific sequences existed in the CP and RTP regions of three BYDV species, respectively, were considered to design three pairs of primers based on the sequence information currently available. Those three pairs of primers could amplified specifically BYDV-GAV, PAV and GPV through RT-PCR The results obtained indicated that the dot blot hybridization based on DIG-labelled cDNA probes were sensitive and specific for the detection each of BYDV-GAV, GPV and PAV. The protocol could detect RNA obtained from as little as 25 ␮g tissue/spot with the probe for GAV, 31.25 ␮g tissue/spot with GPV, and 62.5 ␮g tissue/spot with PAV. The reliability and sensitivity of the method were similar to those reported before (Waterhouse et al., 1986). Dot-blot hybridization using the three probes could either identify the virus species or distinguish single infections from mixed infections. Although RT-PCR makes it possible to detect many virus species of the same genus or family with a single test, and universal primer sets distinguish among more B/CYDV species (Robertson et al., 1991; Malmstorm and Shu, 2004), the tests are not completely suitable under Chinese conditions, because the coat protein genes of GPV and PAV-CN share very low identities with the corresponding species of BYDV (Cheng et al., 1996; Jia et al., 2003; Liu et al., 2007). As long as species-specific probes are generated for each BYDV, a com-

Fig. 6. The number of BYDV-GAV, GPV, and PAV samples, non-reactive samples, and mixed infection samples in 5 agro-ecological areas from which the samples were collected.

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plete assay kit for BYDV identification may be obtained in the near future and will speed up the identification of BYDV in infected plants. Nearly a fifth of the samples gave negative results. It is possible that these plants either harbored other species of B/CYDV or were free entirely of them, the symptoms being due to other causes. Many other pathogens and physiological conditions produce symptoms that are confused easily with those produced by the Barley yellow dwarf virus-indeed, further investigation traced the symptoms to other pathogens. For example, 5 of 6 samples collected in 2004 from Yongning, in Ningxia province, contained WYMV and 3 of 9 samples collected in 2004 from Weinan, in Shaanxi province, were infected by Wheat dwarf virus. Moreover, some samples were infected by Sclerophthora macrospora, a pathogenic fungus. It is also possible that the plants were infected with other species of B/CYDV not yet found in China. However, no species other than BYDV-GAV, GPV, PAV, and RMV have ever been found during the identification and aphid transmission tests on B/CYDV species conducted in this laboratory for nearly 30 years—such a possibility will be explored in further studies. Since the initial report of the technique, dot-blot hybridization using nucleic acid probes has been used by several groups to detect and diagnose BYDVs, and its advantages established (Waterhouse et al., 1986; Habili et al., 1987; Fouly et al., 1992; Lemaire et al., 1995), the principal one being detection without any virus-specific antibodies, which are difficult to produce and in short supply, especially in the less developed countries or regions lacking sophisticated facilities. The high sensitivity and reliability of the molecular hybridization assay described here introduce a significant alternative to serological methods that can be used specifically for detecting BYDV-GAV, PAV, and GPV, the three BYDV species mainly found in China, and detailed information was obtained on the distribution and frequencies of three BYDV species in the different regions during 2004–2006 using the method. The information will increase the understanding of BYDV epidemiology over a larger area and will prove useful in instituting effective control measures. In further studies, total RNA could be extracted from samples and spotted onto nylon membranes at collection sites in collaboration with several local laboratories. Many samples could be collected at the same time and analyzed after collection. Detection kits including DIG-labeled cDNA probes, nylon membranes, and the main reagents can be provided to local laboratories of plant protection services for the detection of BYDV species, an approach with very significant implications for large-scale surveys as well as long-term epidemiological or ecological studies.

Acknowledgments We thank Professor Chenggui Han (China Agricultural University) for supplied the infected wheat samples of WYMV and Mr. Ling Liu and Mengji Cao for technical assistance. Financial support was provided by the National Key Basic Research 973 (2006CB100203 to L.Y. and 2006CB101903 to W.X.F) and Nature Science Foundation of China (NSFC 30470081).

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