Citrus mosaic disease was first reported from India by Murty and Reddy (1975) in sathgudi sweet oranges (Citrus sinensis(L) Osb.) in Andhra Pradesh. It was also reported from northeastern states in khasi mandarins (Citrus reticulataBlanco) (Ahlawat et al., 1985) and in pummelo (Citrus grandis(L) Osb.) in Karnataka (Ahlawat et al., 1996). The incidence of mosaic disease ranges from 10% to 70% in citrus orchards and nurseries and reduction in the yield was up to 77% in trees of 10 years old (Ahlawat et al., 1996). The losses caused by the mosaic disease is apparent in sathgudi sweet orange in Andhra Pradesh and Karnataka because several orchards with trees of 4-8 years old were abandoned since they are no more productive (Ahlawat et al., 1996). The virus was transmitted by grafting and dodder to 13 citrus species and serologically related to four other badnaviruses (Ahlawat et al., 1996). The Citrus Yellow Mosaic Virus (CYMV) is non-enveloped, bacilliform in shape, and virus particles measure approximately 130 x 30 nm (Ahlawat et al., 1996) and contain circular double stranded DNA of 7.5 kb (Huang and Hartung, 2001).

Badnaviruses are mostly transmitted by mealybugs (family Pseudococcidae) and some are also transmitted through seed and pollen (Lockhart and Olszewski, 1994). An improved method for isolation of DNA from virus infected plants was standardized and a PCR technique was developed for the detection of CYMV from field and glasshouse isolates (Barnawal et al., 2003). Since citrus is vegetatively propagated, and the disease is transmitted through grafting , dodder and sap, it is essential to have sensitive reliable indexing method for large-scale screening of bud wood banks for CYMV. Detection of badnaviruses is often difficult by ELISA because the concentration of virus is very low in host (Lockhart and Olszewski, 1994). Keeping this in mind we developed sensitive and reliable indexing methods for large- scale detection of this economically important virus by nucleic acid hybridization, using both radioactive and non-radioactive probes.

The bud sticks collected from sathgudi sweet orange from Kodur (Andhra Pradesh) were wedge grafted on to six-months-old pummelo seedlings. The virus culture was maintained on pummelo seedlings in an insect-proof glasshouse. The probe used for detection of CYMV is cloned 2.0 kb virus-specific fragment in pUC18 at Hind III site named as pCMBH-2.0 (Reddy et al., 2007).

The total DNA from the leaves of infected and healthy plants were isolated by CTAB method (Dellaporta et al., 1983) with minor modifications. 500 mg of leaf material was grounded to fine powder in liquid nitrogen using pestle and mortar and added two volumes of extraction buffer (2% CTAB, 1.4 M NaCl, 100 mM Tris-Hcl (pH 8.0), 10 mM EDTA, 0.5% Na₂SO₃). The homogenate was incubated at 65 ^oC for 1 h with occasional shaking and centrifuged at 12,000 rpm in Sorvall SS-34 rotor for 30 min. at 20 ^oC. The supernatant was transferred to fresh sterile tube and mixed with equal amount of Tris saturated phenol:chloroform:iso-amyl alcohol (25:24:1) and centrifuged at 10,000 rpm for 10 min. The aqueous phase was transferred to fresh tube and finally the DNA was precipitated with 0.8 volumes of isopropanol. The DNA pellets were washed with 70% ethanol, air-dried and dissolved in 50 mL of TE buffer (10 mM Tris-Hcl, 1 mM EDTA) and stored at 20 ^oC for further use.

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