Association of a Disease Complex Involving a Begomovirus, DNA 1 and a Distinct DNA Beta with Leaf Curl Disease of Okra in Pakistan

Okra leaf curl disease (OLCD), characterized by either upward or downward leaf curl and stunted plant growth, is one of the major diseases of okra (Hibiscus esculentis L.) in Pakistan. OLCD is transmitted by the whitefly Bemisia tabaci and is suspected of being associated with a whitefly-transmitted geminivirus (Genus Begomovirus). Total DNAs isolated from both symptomatic and healthy okra plants collected from several locations in Pakistan were resolved on agarose gels and blotted to nylon membranes. A full-length DNA A clone of Cotton leaf curl virus (CLCuV) from Pakistan (2) was labeled with 32PdCTP and used as a probe at medium stringency. The probe detected the presence of characteristic geminivirus DNA forms in infected plants, while no hybridization was observed to healthy plant extracts, confirming the association of a begomovirus with OLCD. Degenerate oligonucleotide primers based on conserved sequences of DNA B components of begomoviruses were used in PCR for the detection of a potential DNA B (3). No amplification was observed with these primers from okra plants, while amplification of a product of expected size was obtained from plants infected with African cassava mosaic virus, suggesting the lack of a genomic component equivalent to DNA B. We have reported previously that monopartite begomoviruses on cotton and Ageratum conyzoides in Pakistan are associated with a disease complex involving a DNA component termed DNA 1, which shows homology to components of nanoviruses that encode the replication-associated protein (2). Recently, another molecule, DNA beta, has been identified, associated with Ageratum yellow vein disease from Singapore (4) and with cotton leaf curl disease (CLCuD) from Pakistan (1). These molecules are DNAs satellite and are essential for the development of typical disease symptoms in their respective hosts. Duplicate blots were probed for the presence of DNAs homologous to DNA 1 and DNA beta (using full-length clones of these molecules isolated from CLCuD originating from Pakistan [1,2]) and washed at medium stringency. The probes detected bands hybridizing to DNA 1 in extracts from infected okra plants but not DNA beta. No hybridizing bands were detected for either probe in extracts from healthy okra. A pair of primers, designed to conserved sequences in DNA beta molecules (4), were used in PCR for the amplification of DNA beta from symptomatic plants. The use of these primers amplified a product of the expected size (approximately 1.35 kb) from extracts of infected okra plants. The amplified DNA was cloned in TA cloning vector and labeled with 32PdCTP. The use of this as a probe detected the presence of a hybridizing band in infected okra plants, while no signal was observed in extracts from cotton plants showing symptoms of CLCuD. These results show that OLCD in Pakistan is associated with a DNA beta molecule that is distinct from that reported on cotton and Ageratum. In particular, the DNA beta of CLCuD and OLCD originating from Pakistan are sufficiently diverse not to cross-hybridize under the conditions used here, and are most likely different disease complexes. To our knowledge this is the first report of the association of a whitefly-transmitted begomovirus/DNA 1/DNA beta complex with okra leaf curl disease. References: (1) R. W. Briddon et al. Virology, 2001 (In press). (2) S. Mansoor et al. Virology 259:190, 1999. (3) M R. Rojas et al. Plant Dis. 77: 340, 1993. (4) K. Saunders et al. PNAS 97:6890, 2000.