The breakdown of resistance in cotton to cotton leaf curl disease in Pakistan

Cotton leaf curl disease (CLCuD), a devastating disorder of cotton in Pakistan, is caused by a whitefly-transmitted begomovirus (Cotton leaf curl virus; CLCuV) that requires a satellite DNA b to cause disease symptoms (Mansoor et al., 1993; Briddon et al., 2001). CLCuD-resistant cotton varieties, in which no virus can be detected, have been developed through conventional breeding (Rehman et al., 2002). During the 2001 growing season symptoms of CLCuD (Fig. 1) were observed on all hitherto resistant varieties at Burewala, District Vehari and by 2002 disease symptoms were seen throughout the district.

To determine if a resistance breaking strain of CLCuV had arisen, resistant and susceptible varieties were grown in the field at NIBGE (Faisalabad) and at the Cotton Research Station (Vehari). Plants of six commercial virus resistant varieties (CIM 448, CIM 443, CIM 446, CIM 473, CIM 435 and FH 900) showed no disease symptoms at Faisalabad while susceptible varieties S-12 and CIM70 had symptoms typical of CLCuD. At Vehari plants of the same six resistant varieties showed between 15-50 % infection, while the two susceptible varieties were all infected. Scions of CLCuD-affected resistant varieties, collected from Vehari, were grafted onto ten plants of each resistant genotype at NIBGE. This resulted in disease symptoms on 20-40 % of plants, confirming a breakdown of resistance. To identity the resistance breaking virus, nucleic acid was extracted from both symptomatic and asymptomatic plants collected at both sites. Samples were Southern blotted and probed with a biotinylated DNA A clone of CLCuV. The probe detected both the ss and ds DNA forms characteristic of begomoviruses, confirming the association of a begomovirus with the disease. Universal primers for DNA b of CLCuV were used to amplify DNA b from symptomatic leaves collected from resistant varieties in the Vehari area and the PCR product from one location was cloned in a T/A cloning vector (Fermentas). Since CLCuV DNA b is specific to CLCuV (Briddon et al., 2003), a DNA b cloned from cotton plants of resistant varieties showing symptoms of CLCuD in the Burewala area was used as a disease specific probe in Southern blot hybridizations. The probe hybridized only with DNA extracted from symptomatic cotton plants while no signal was detected from a tomato plant (Lycopersicon esculentum) that was previously shown to be associated with a DNA b distinct from that associated with CLCuV (Briddon et al., 2003). Samples collected from both locations hybridized with this probe (Fig. 2 lower panel). A duplicate blot was probed with a previously reported CLCuV DNA b (Briddon et al., 2001) and this resulted in a similar pattern of hybridization (Fig 2 upper panel). Based on the data presented here, we concluded that the plants of resistant varieties were infected with CLCuV (Briddon et al., 2001). These results strongly suggest the emergence of a resistance-breaking strain of CLCuV in Pakistan.