The Journal of Cotton Science 9:175–181 (2005) 175 http://journal.cotton.org, © The Cotton Foundation 2005 PLANT PATHOLOGY AND NEMATOLOGY Resistance to Cotton Leaf Curl Virus (CLCuV) in a Mutant Cotton Line Khalid Pervaiz Akhtar*, M. Aslam, M. Ahsanul Haq, Farhat Fatima Jamil, Azeem Iqbal Khan, and M. Tanvir Elahi ABSTRACT The disease was first observed in Pakistan near Multan on a few cotton plants in 1967. At that time, Cotton leaf curl virus (CLCuV) is one of the the disease was of minor importance and it did not major biotic constraints of cotton production in attract serious attention. In 1988, only 60 hectares Pakistan. Cultivation of resistant cotton genotypes in the Multan district were damaged. Since 1988, is the most effective method of reducing yield the geographic distribution of CLCuV has increased losses due to CLCuV. PIM-76-8/5 is a new CL- greatly (Mahmood, 1999), and more than 7.7 million CuV-resistant line developed through the use of bales of cotton have been lost to CLCuV from 1988 induced mutation. It exhibited a highly resistant through 2002 (Ahmad et al., 2002). response when artificially inoculated by grafting Development of a CLCuV-resistant cultivar is the and produced yields significantly greater than the most promising control option (Akhtar et al., 2003). susceptible cultivar S-12. PIM-76-8/5 was field The Nuclear Institute for Agriculture and Biology immune when naturally infected by the whitefly (NIAB) in Faisalabad, Pakistan, has initiated research (Bemisia tabaci Genn.), the vector of CLCuV. At to develop high yielding, CLCuV-resistant cotton a few locations where a new strain of CLCuV has cultivars with desirable fiber characteristics through emerged and all of the previously developed resis- the use of induced mutations. The CLCuV-resistant tant lines are now highly susceptible, PIM-76-8/5 mutant PIM-76-8 was the result of an interspecific gave a moderate to highly susceptible response. At cross between NIAB-78 and REBA-288. The male locations with high levels of whitefly infestation parent pollen was irradiated with 10Gy of gamma rays. or with artificial inoculation through grafting in On average, PIM-76-8 produced yields 28.2 % higher this study, PIM-76-8/5 exhibited a high level of than the standard CLCuV-resistant cultivar CIM-443 resistance against the old strain of CLCuV. in micro-yield trials conducted at NIAB during 1998 and 1999 (Aslam and Elahi, 2000). otton leaf curl virus (CLCuV) is one of the This study was conducted to determine the level Cmost destructive diseases of cotton (Gossypium of resistance of PIM-76-8 to CLCuV under field hirsutum L.) in the Punjab area of Pakistan (Nelson conditions at locations where CLCuV severity is et al., 1998). CLCuV is in the family Geminivirus, normally very high. Single-plant progeny rows were genus Begomovirus (Hameed et al., 1994) and is evaluated for resistance through graft inoculation transmitted by the whitefly (Bemisia tabaci Genn.) with CLCuV-infected plant material collected from (Nadeem et al., 1997; Nelson et al., 1998). The virus different locations. has single-stranded circular DNA (ssDNA) with two geminate particles. CLCuV disease is characterized MATERIALS AND METHODS by upward or downward curling of leaves. The leaf veins are thickened on the underside of leaves. In Field evaluations. The response of 12- to 16- some infections, one or more cup-shaped outgrowths week-old plants of PIM-76-8 to infection by CLCuV called “enations” appear on the underside of the leaf. under different environments in 26 producer fields When CLCuV is severe, infected plants are stunted in the cotton growing area of Punjab province was (Hussain, et al., 1991; Brown, 2001). studied during August of 2000 and 2001. All of these locations were known for high incidence of CLCuV infection. The reactions of three resistant cultivars K. P. Akhtar, M. Aslam, M. A. Haq, F.F. Jamil, A.I. Khan, and (CIM-473, CIM-446, and FH-900) and two suscep- M.T. Elahi, Nuclear Institute for Agriculture and Biology, P. tible cultivars (NIAB-Karshmia and NIAB-78) were O. Box-128, Jang Road, Faisalabad, Pakistan. also determined. Fifty to 300 plants (depending on * Corresponding author: [email protected] the area under cultivation of the test cultivar) in each Akhtar ET AL.: Resistance to Cotton LEAF CURL VIRUS 176 location were randomly chosen. None of the plants long growing tip was detached from a diseased were sprayed for whitefly control. Disease index (%) plant. A cut similar to the one made on the test plant and reaction of the cultivars was determined using was made on this branch, and the corresponding the disease scale described by Akhtar and Khan cut surfaces were brought together and tied with (2002) (Table 1). parafilm. This stem was then placed in a 2-cm dia., The response of six single plant progeny rows of 16-cm long test tube containing distilled water. The PIM-76-8 (PIM-76-8/6, PIM-76-8/7, PIM-76-8/18, distilled water was changed daily for 5 d. After 5 d PIM-76-8/20, PIM-76-8/38, and PIM-76-8/5) and a the tubes were removed, and plants were observed resistant (CIM-473) and susceptible (S-12) standard to daily for symptom development. CLCuV using natural inoculation by whiteflies were In another study, four to six seeds of PIM-76- determined during September in 2000 and 2001 at the 8 collected from different locations were sown in Nuclear Institute for Agriculture and Biology (NIAB) earthen pots under insect-free conditions during in Faisalabad. Disease index (%) and reaction of the 2000-01. The commercial cultivars CIM-473 and mutant lines and cultivars was recorded. No insecti- S-12 were included as resistant and susceptible cides were applied to control the whiteflies. Similar controls, respectively. Plants were thinned to one studies were performed with single plant progeny rows per pot 2 wk after germination and grafted for of PIM-76-8/5 during 2001-02 and 2002-03 at a cot- artificial inoculation. Clean tap water was used to ton experimental area of NIAB. Data from National irrigate the young seedlings throughout the study. Coordinated Varietal Trials (NCVT) and Director The percentage of successful grafts, percentage of General Research trials (DGR) were also collected disease transmission, mean latent period (average for PIM-76-8/5 (NIAB-98). time required for first disease symptoms to appear Graft inoculation studies. CLCuV inoculum after grafting), and average disease severity at 90 d for all graft inoculation studies was collected from after grafting were recorded. naturally-infected cotton plants exhibiting charac- In a third study, six single plant progeny rows of teristic leaf curl symptoms of CLCuV. The virus was PIM-76-8 (PIM-76-8/6, PIM-76-8/7, PIM-76-8/18, maintained by grafting infected plants onto plants PIM-76-8/20, PIM-76-8/38, and PIM-76-8/5) were grown in a greenhouse of the commercial cultivar graft-inoculated in the field during the fall of 2000- S-12, which is susceptible to CLCuV. Grafting was 01 to determine their reaction to CLCuV. CIM-473 performed following the bottle leaf grafting method and S-12 were included as resistant and susceptible described by Akhtar et al. (2002c). Six-week-old standards, respectively. Ten plants in each replica- plants were selected for graft-inoculation. A 1- to tion were covered with insect proof cages made of 2-cm long and 0.1- to 0.2-cm deep cut was made very fine meshed cloth. Six weeks after emergence, on the stem at the 2nd internode from the top of one plant under each cage was inoculated by graft- the plant. A CLCuV-infected branch with a 20-cm ing. During 2001-02, seven plants of PIM-76-8/5 Table 1. Rating scale for cotton leaf curl virus symptoms Symptoms Disease rating y Disease index (%) z Disease reaction Absence of symptoms. 0 0 Immune Thickening of a few small veins or the presence of 1 0.1- 1 Highly resistant leaf enations on 10 or fewer leaves of a plant. Thickening of a small group of veins. 2 1.1- 5 Resistant Thickening of all veins but no leaf curling. 3 5.1-10 Moderately resistant Severe vein thickening and leaf curling on the top 4 10.1 – 15 Moderately susceptible third of the plant. Severe vein thickening and leaf curling on the half 5 15.1 – 20 Susceptible of the plant. Severe vein thickening, leaf curling, and stunting of 6 >20 Highly susceptible the plant with reduced fruit production. y Disease ratings that include enations are marked with an “E”. z The percentage disease index was calculated as follows: (sum of all disease ratings/total # of plants) x 16.66. JOURNAL OF COTTON SCIENCE, Volume 9, Issue 2, 2005 177 were graft-inoculated using the same methodology RESULTS under field conditions. Data were recorded on the percentage of successful grafts, percentage of dis- Field evaluations. Response of PIM-76-8 to in- ease transmission, mean latent period (average time fection by CLCuV was recorded in 26 farmer’s fields required for first disease symptom appearance after located in the Punjab during 2000-01. PIM-76-8 was grafting), and average disease severity at 90 days field immune (no plants with symptoms) at 8 loca- after grafting. Disease severity and reaction of test tions, highly resistant at 15 locations, and resistant at lines and cultivars was recorded. 3 locations (Table 2). The susceptible control exhibited Table 2. Cotton leaf curl development in PIM-76-8 under natural infestation by the whitefly (B.