Association of a Monopartite Begomovirus Producing Subgenomic DNA and a Distinct DNA Beta on Croton bonplandianus Showing Yellow Vein Symptoms in Pakistan

Abstract

The recent discovery that monopartite begomoviruses on ageratum and cotton essentially require a DNA satellite called DNA β (2,4) is leading to identification of several other hosts that have similar disease complexes. A weed species (Croton bonplandianus) belonging to the family Euphorbiaceae is one such example. C. bonplandianus is widely distributed on wastelands throughout the Punjab Province in Pakistan. It very often shows yellow vein symptoms indicating infection by a begomovirus. To detect a begomovirus, both symptomatic and asymptomatic plants were collected from several widely separated locations in the Punjab Province. Total DNA was isolated from these samples by the cetyltrimethylammoniumbromide (CTAB) method, resolved in an agarose gel, and blotted on a nylon membrane (2). A full-length clone of DNA A of Cotton leaf curl virus (CLCuV) labeled with 32PdCTP was used as a probe in Southern hybridization (2). The probe detected hybridizing bands only in symptomatic plants, confirming the presence of a begomovirus. In addition to hybridizing bands of the expected sizes, smaller bands were also detected, suggesting the presence of subgenomic molecules derived from DNA A. Universal polymerase chain reaction (PCR) primers for dicot-infecting geminiviruses (1) were used in PCR for amplification of DNA A of the begomovirus associated with the disease. The use of these primers in PCR was expected to result in amplification of full-length DNA A. In addition to a product of the expected size (2.7 to 2.8 kb), another product of approximately 1.4 kb was amplified. The presence of subgenomic DNAs that are derived from DNA A is an indicator of the monopartite nature of begomoviruses, because in bipartite begomoviruses subgenomic DNAs are derived solely from DNA B. The presence of a DNA β, a DNA satellite associated with certain monopartite begomoviruses, was suspected because of symptoms and the possible monopartite nature of the virus. Universal primers for amplification of DNA β (3) were used in PCR for amplification of a putative DNA β. The PCR reaction yielded a product of expected size (≈1.4 kb). A probe from the amplified product was made by the oligolabeling method. The probe detected hybridizing bands in all symptomatic samples collected from three locations, confirming the association of a DNA β with the disease. A duplicate blot when hybridized with a DNA β associated with ageratum yellow vein disease did not hybridize to these samples. These results confirm that yellow vein disease on this weed is associated with a monopartite begomovirus and a distinct DNA β. References: (1) R. W. Briddon et al. Mol. Biotechnol. 1:202, 1994. (2) R. W. Briddon et al. Virology 285:234, 2001. (3) R. W. Briddon et al. Mol. Biotechnol. In press. (4) K. Saunders et al. Proc. Natl. Acad. Sci. U S A 97:6890, 2000.