Partial resistance of a cotton mutant to Cotton leaf curl Burewala virus

Cotton leaf curl disease (CLCuD), caused by Cotton leaf curl Burewala virus (CLCuBV), is a major constraint for a successful production of cotton in Pakistan. All the available cotton cultivars are susceptible to this virus. Breeding for resistance to CLCuBV is the best tactic to reduce economic losses caused by this virus. In the absence of a resistant source the present investigations were carried out to find out CLCuBV-resistant genotypes by mutagenization. NIAB-111 (female parent) was crossed separately with two male parents, NIAB-999 and CIM-499. The resulting F1 seeds were gamma irradiated. Resistance to CLCuBV was evaluated by visual symptom scoring in the f ield conditions and in nethouse/greenhouse using whitefly and graft inoculations. Out of 20 mutants tested in M5 generation, one, mutant M-112-59/22 showed partial resistance to CLCuBV, as concluded from its low severity index (SI) of 2.3 and percent disease index (PDI) of 20. M-112-59/22 consistently expressed resistance to CLCuBV in the normal cotton growing season, while it showed a more moderate resistance response when sown late in the field, or under greenhouse conditions following inoculation by whiteflies (SI = 3.3) or graft inoculation (SI= 3.4). The yield per plant of M-112-59/22 was higher than its parents with desirable fiber characteristics even under conducive disease development conditions. These results show that mutant M-112-59/22 is a CLCuBV partially resistant source when yield, fiber quality and response to virus infection are collectively taken into consideration. Additional key words: begomovirus; CLCuBV; CLCuD; Gossypium hirsutum; partially resistant mutant.

Since 1988, cotton (Gossypium hirsutum L.) in Pakistan is under constant threat of cotton leaf curl disease (CLCuD).A combination of distinct whitefly (Bemisia tabaci Genn.) transmitted begomoviruses has been shown to be associated with CLCuD (Briddon, 2003).Begomoviruses associated with CLCuD are characterized by twined isometric particles, 18-20 nm in diameter and 30 nm long, containing a circular single stranded DNA molecule.About seven species of begomoviruses have been reported so far and five of these are in Pakistan and one each in India and Sudan (Amin et al., 2006;Sharma and Rishi, 2007), all requiring a recently identified symptom modulating single stranded betasatellite component.An additional, satellite-like component, alfasatellite, is invariably found in infected plants, although it is not required for disease development (Briddon, 2003).Recently, a recombinant begomovirus derived from Cotton leaf curl Multan virus (CLCuMV) and Cotton leaf curl Kokhran virus (CLCuKV) has been associated with the resistance breaking strain of CLCuD present in Pakistan since 2001.Moreover, recent sequence analysis of viruses associated with the resistance breaking complex in this country revealed that only this recombinant begomovirus type is prevalent, in contrast to the situation before the appearance of the resistance breaking strain (Amin et al., 2006;Amrao et al., 2007).This recombinant virus was named as Cotton leaf curl Burewala virus (CLCuBV), which is now prevalent in cotton-growing areas of Pakistan.
Attempts to manage CLCuD by the control of inoculum reservoirs and vector populations have been ineffective particularly during high inocula pressures.Breeding resistant varieties is one of the best ways to combat CLCuD.Since its outbreak in Pakistan, there have been considerable efforts to develop CLCuDresistant varieties using conventional and mutation breeding (Ali, 1997;Awan et al., 1998;Akhtar et al., 2000;2002a,b;2004).Studies showed that disease was efficiently managed by developing resistant cotton varieties, but the recent emergence of resistance-breaking strain of virus breakdown the resistance and all previously resistant varieties become susceptible (Mahmood et al., 2003;Akhtar et al., 2008Akhtar et al., , 2009Akhtar et al., , 2010)).Therefore, we planned this study to search for cotton plants resistant to this new recombinant virus, CLCuBV, by mutagenization.
Allotetraploid Gossypium hirsutum L. NIAB-111 (female parent) was crossed with NIAB-999 (male parent) and CIM-499 (male parent) to produce M-112 and M-219 respectively.F 1 seeds were gamma irradiated and M 1 was sown during 2002 crop season.The parents were selected on the basis of their resistant response to CLCuMV (Akhtar et al., 2008), high yield, heat tolerance and good boll size.Resistant mutants from M 2 were selected on the basis of their field response.From M 3 to M 7 generations, selections were made according to their degree of resistance to CLCuD.However, M 5 to M 7 generations were evaluated under the present study.
Field assessments were made between 2006 to 2008 growing season at Nuclear Institute for Agriculture and Biology (NIAB), Faisalabad (hot spot for CLCuBV), Pakistan.Seeds were sown in field on 18 th and 28 th of May in 2006 and 2007 respectively.During 2008 seeds were sown on 20 th May and 9 th June with normal plant to plant and row to row distance.Normal agronomic practices were followed to keep the crop in good conditions.However no plant protection measures were applied against whiteflies in order to have the maximum inoculum pressure during the experiment.Data for CLCuBV were recorded following the rating system described in Table 1 to calculate severity index (SI), percent disease index (PDI) and response of genotypes.Individual symptomatic plant ratings (following Table 1) for each genotype were added and divided by the number of infected plants to calculate the corresponding SI.The PDI was calculated using the following formula: Sum of all disease ratings 100 PDI = --------------× --Total plants observed 6* * = maximum disease severity rating The sum of all disease ratings was obtained by multiplying the disease severity rating with the corresponding number of plants.
The inoculum of CLCuBV for graft inoculation and whitefly transmission was taken from naturally infected cotton plants in experimental area of NIAB Faisalabad and maintained in CIM-499 (positive control for CLCuBV) by grafting in the glasshouse.
Five to six weeks old plants grown in glasshouse, 10 of each test entry were graft-inoculated with CLCuBV following the method described by Akhtar et al. (2002c).Plants were observed daily for symptom development.Data were recorded on the percentage of successful grafts, percentage of disease transmission, mean latent period (average time required for disease symptom appearance after grafting), and average SI after 90 days of grafting using the rating scale described in Table 1.
For whitefly transmission, adult whiteflies collected from cotton fields were provided a 72-h acquisition access period (AAP) on CLCuBV-infected cotton plants.A total of 10 potted cotton plants (5-6 weeks old) per test entry (5 plants per cage) were inoculated using 100 potentially viruliferous whiteflies per plant.After 72-h inoculation access period (IAP) plants were sprayed with an insecticide to kill whiteflies.The inoculated plants were transferred into a net-house under insect free conditions.This experiment was conducted during the normal cotton growing season to provide natural environmental conditions.Plants were observed daily for symptom development.Data were recorded on the percentage of disease transmission, mean latent period, and average SI after 90 days of transmission using the rating scale described in Table 1.
At maturity 10 randomly selected plants of each cotton genotype were hand-picked from the field.The seed cotton for each sample was weighted, cleaned and then ginning of seed cotton was made by the standard miniature roller gin.The lint obtained was weighed and the ginning out-turn (GOT) percentage was calculated by the formula [(lint weight/seed weight.)× 100], according to the standard technique of ASTM Committee (1997).Yield components consisting of number of bolls per plant and average boll weight was also determined from these plants.Fiber length was measured by high volume instrumentation (HVI 900), and fiber fineness with the help of Sheffield micronaire instrument, according to standard tests methods (ASTM, 1997).
Twenty mutant cotton plants, one female parent and two male parents listed in Complete absence of symptoms and virus can not be detected in plant tissues using molecular techniques.
Complete absence of symptoms but virus can be detected in plant tissues using molecular techniques.OR Thickening of few small scattered veins or only presence of leaf enations on one or few leaves of a plant observed after careful observations.
Thickening of small group of veins, no leaf curling, no reduction in leaf size and boll setting.
Thickening of all veins, minor leaf curling & deformity of internode with minor reduction in leaf size but no reduction in boll setting.
Severe vein thickening, moderate leaf curling followed by minor deformity of internodes and minor reduction in leaf size and boll setting.
Severe vein thickening, moderate leaf curling & deformity of internodes with moderate reduction in leaf size and boll setting followed by moderate stunting.
Severe vein thickening, leaf curling, reduction in leaf size, deformed internodes and stunting of the plant with no or few boll setting.2).The average number of bolls per plant for M-112-59/22 was 82 with an average yield per plant of 218.42 g (Table 2).
During 2008 the response of M-112-59/22 was again compared with parents in the field at two different sowing dates (Table 2).M-112-59/22 was highly resistant when planted during the normal sowing season, while the other varieties were highly susceptible to susceptible.In the late sown crop, all plants of male and female parents were infected, while 30% plants of M-112-59/22 were free from the disease 40-50 days after germination.When sown late, M-112-59/22 was moderately resistant with 26.10 PDI, while parents NIAB-111 and NIAB-999 were highly susceptible with PDI of 92.93 and 97.24, respectively.However, M-112-59/22 showed an average SI of 1.7 as compared to 5.6-5.8 for the parents under conducive conditions.At both sowing dates M-112-59/22 showed higher number of bolls (84 and 68) and higher average yield per plant (220 g and 167 g) than the parents.Average boll weight, GOT percentage, fiber length and fiber fineness of M-112-59/22 were very similar for both the sowing dates.
The response of M-112-59/22 to inoculation through grafting with CLCuBV was compared to that of NIAB-111 and NIAB-999.All the tested plants showed symp-toms, indicating that the success of grafting and disease transmission was 100%.The data presented in Table 3 show high variations in disease severity of the cotton genotypes tested.M-112-59/22 showed attenuated symptoms varying between SI 3 to 4 with an average SI of 3.4, being ranked as moderately resistant.NIAB-111 was defined as susceptible with average SI of 5.3, while NIAB-999 was highly susceptible with average SIs of 6.0.The first disease symptoms started in the parent NIAB-999 as minute vein thickening after 9 days of inoculation (DPI) and culminating in severe vein thickening, leaf curling and severe stunting after 15 DPI.Concurrently, NIAB-111 showed more moderate symptoms but the symptoms appearing in M-112-59/22 were clearly milder.However, the latent period did not differ greatly as the disease started after 12, 11 and 9 DPI on M-112-59/22, NIAB-111 and NIAB-999, respectively.
Ten plants each from M-112-59/22, NIAB-999 and NIAB-111 were also compared for their response to
Breeding of CLCuD-resistant cotton with high yield and appropriate fiber characteristics is the most important issue.Considering the increasing intensity of CLCuD during the past years, identification of CLCuD resistant cotton varieties after mutagenesis was initiated at NIAB, Faisalabad, Pakistan during 1995 (Awan et al., 1998).Mutation breeding is a complimentary approach to the conventional breeding which provides an opportunity to improve a crop cultivar for a particular trait without disrupting the genotype or to break undesirable linkage between existing genes (Awan et al., 1998).By this technique a number of CLCuMV resistant mutants were developed (Awan et al., 1998;Akhtar et al., 2000Akhtar et al., , 2002bAkhtar et al., , 2004)).During 2001 a resistance breaking strain appeared and all the resistant germplasm became susceptible (Akhtar et al., 2002a(Akhtar et al., , 2008(Akhtar et al., , 2010;;Mansoor et al., 2003).
The present study showed that resistance response of mutant M-112-59/22 was better than that of its parents when evaluated through different inoculation techniques.In the f ield tests M-112-59/22 showed a better resistance response and a higher production with acceptable fiber characteristics than the parents.The parents were susceptible to highly susceptible to CLCuBV over the last three seasons, suggesting that the inoculum level was appropriate to cause severe disease.Field screening is the most commonly used method for assaying CLCuD resistance.But this screening technique sometimes produces misleading results due to spatial and temporal variations in inoculum levels due to the effect of environmental conditions, vector host preference and possible host resistance to the vector (Rahman et al., 2005;Akhtar et al., 2010).These ambiguities were avoided by using graft inoculation and whitefly transmission in the greenhouse.M-112-59/22 showed similar level of severity with infection type range (ITR) of 3-4 using both transmission methods which supported the f ield f indings.However, this mutant showed a higher average SI value in these tests as compared to the field, but still lower than the parents.The majority of plants of M-112-59/22 have shown an average SI of 3, which means that this mutant can produce good yield even under high disease pressure.
Using disease symptom development, yield and fiber quality parameters as the indicator of resistance M-112-59/22 is considered to be a CLCuBV partial resistant mutant that has medium boll size, early maturity, thin leaf, normal foliage, medium long fine fiber and medium short to medium sympodial (data not shown).It can be directly used in the field to reduce the losses caused by CLCuBV.The host reaction that reduces the same kind of epidemics has previously been reported by many workers for other viral pathosystems (Padgett et al., 1990;Buiel and Parlevliet, 1995;Dintinger et al., 2005;Delatte et al., 2006).
In conclusion it is important to note that the high level of resistance to CLCuBV was not observed in the mutants tested here.Due to the acceptable agronomic characteristics and partial resistance to CLCuBV, M-112-59/22 would be useful for managing the present epidemics of CLCuD and will be an essential tool in the hands of the breeders for the development and selection of new resistant genotypes.

Table 1 .
Disease scale for rating of cotton leaf curl disease (CLCuD)

Table 2 .
Partial resistance of cotton mutant to CLCuBV1101 Field response of cotton mutants to CLCuBV infection from 2006 to 2008 a Av.SI: average severity index.b PDI: percentage disease index.c DR: disease response; HR: highly resistant; R: resistant; MR: moderately resistant; MS: moderately susceptible; S: susceptible; HS: highly susceptible.d Female parent.e Male parent.

Table 3 .
Response of cotton mutant M-112-59/22 to CLCuBV inoculated through grafting in glasshouse or by whiteflies Bemisia tabaci under net-house conditions