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The breakdown of resistance in cotton to cotton leaf
curl disease in Pakistan
S. Mansoor1*, I. Amin1, S. Iram1,
M. Hussain1, Y. Zafar1, K. A. Malik1
and R.W. Briddon2
1 National Institute for Biotechnology and Genetic
Engineering (NIBGE), Faisalabad, Pakistan.
2 Department of Disease and Stress Biology, The John Innes
Centre, Norwich, United Kingdom
*smansoor@nibge.org
Accepted for publication 06/03/03
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.
Figure 1. Symptoms of cotton leaf curl disease at Burewala, Pakistan on
resistant variety FH 900. Thick dark veins and leaf-like outgrowths or
enations can be seen on the main vein
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Figure 2. Southern blot hybridization using CLCuV DNA b as
specific probes. Upper panel: a CLCuV DNA b probe (Briddon et
al., 2001). Lower panel: a CLCuD DNA b cloned from infected
plants of resistant varieties collected from the Burewala area.
Lane 1: Symptomed cotton var. S12 (susceptible), Faisalabad; Lane
2: Symptomless cotton Faisalabad; Lane 3: CLCuV infected tobacco,
Faisalabad; Lane 4: Graft inoculated cotton of resistant variety
Faisalabad; Lane 5: Tomato with leaf curl symptoms; Lane 6-10:
Resistant cotton plants with symptoms of CLCuD from different
sites in Vehari district.
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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. |
References
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