Cotton ( Gossypium hirsutum L.) is an important crop globally for its diverse uses ( Zhang et al. , 2008 ). It is important for fiber, oil, biofuel production and also feedstock industry ( De-Sousa et al. , 2015 ). It is also called as white gold for its importance as cash and industrial crop ( Iqbal et al. , 2005 ). China, India, USA and Pakistan are major cotton producing countries. A large number of people throughout the world are involved in different aspects of cotton production, cotton products processing, and cotton products trade ( Zhang et al. , 2008 ). In Pakistan, cotton accounts for more than 50% of total foreign export earnings ( Anonymous, 2017 ).

Cotton production is faced with a number of biotic and abiotic stresses. Among these production constraints, CLCuVD (cotton leaf curl virus disease) severely affects cotton production world-wide ( Farooq et al. , 2011 ; Hashmi et al. , 2011 ). This disease is caused by a virus known as Cotton leaf curl virus (CLCuV). CLCuV belongs to genus Begomovirus and family Geminiviridae . It was noticed for the first time in Nigeria ( Farquharson, 1912 ), and the first symptoms of CLCuVD were observed in Pakistan during 1967 ( Hussain & Ali, 1975 ). In Pakistan, CLCuVD appeared in epidemic form after 1988 and heavy cotton production losses occurred in 1992 due to vast cotton cropped area affected by CLCuVD infestation. In 1992, world cotton production was also affected badly due to CLCuVD infestation. Cotton breeders started efforts to find CLCuVD resistant germplasm. Conventional breeding efforts were fruitful and soon CLCuVD resistant varieties of G. hirsutum were developed.

Rate of evolution in CLCuV genome is relatively high. In 2001, a new strain of this virus was noticed in Burewala, district Vehari, Punjab, Pakistan. This strain was named CLCuVB ( Cotton leaf curl virus Burewala). All resistant genotypes of upland cotton became susceptible to this new viral strain ( Mansoor et al. , 2003 ). Nowadays, there is no report of any available upland cotton cultivar resistant to CLCuVB attack. It has direct social and economic concerns. In Pakistan, approx. 30% yield losses per year are caused due to CLCuVD ( Briddon et al. , 2000 ; Rahman et al. , 2014 ; Hassan et al. , 2016 ). In past, this disease was reported in Pakistan and India. But, it has spread to other important cotton growing areas of the world also such as southern China ( Mao et al. , 2008 ; Cai et al. , 2010 ).

Plants affected by CLCuVD show a complex of symptoms including downward cupping of younger leaves, veins swelling and thickening, and leaf margins upward or downward curling. Frequently, there is emergence of outgrowths, resembling small leaves, on the lower side of leaves. These outgrowths are known as ‘enations’ ( Akhtar et al. , 2008 ). In severely affected plants, yield is adversely low and poor-quality fiber is produced ( Akhtar et al. , 2009 ). Fortunately, CLCuVD is not transmitted through seed, but has the capacity to survive in different hosts ( Khan & Ahmad, 2005 ). Because its vector is whitefly ( Bemisia tabaci Genn.), an approach to limit the infection is to control the population density of this insect. But, this is not a practically feasible solution ( Holt et al. , 1999 ). There may be chances of evolution of pesticide resistance in whiteflies and also environmental concerns about heavy use of pesticides ( Palumbo et al. , 2001 ). Search for resistant sources is the most desirable solution to handle a disease stress and it is an environment friendly option ( Hogenboom, 1993 ).

Cuticular wax, an important constituent of plant cuticle, has been found to give resistance to plants against a number of biotic and abiotic stresses ( Smith, 1999 ; Jeffree, 2006 ; Kosma et al. , 2010 ; Bourdenx et al. , 2011 ; Weidenbach et al. , 2014 ). Cuticular wax is reported to inhibit attack of different pathogens on various plant species ( Martin et al. , 1957 ; Johnston & Sproston, 1965 ; Heather, 1967 ; Blakeman & Sztejnberg, 1973 ; Alcerito et al. , 2002 ; Kosma et al. , 2010 ; Yin et al. , 2011 ; Weidenbach et al. , 2014 ). Thinning of wax layer on leaves is found to promote susceptibility to stress conditions ( Rawlinson et al. , 1978 ; El-Otmani & Coggins, 1985a , b ).

This research project was designed to investigate the role of cuticular wax content (WC) in resisting CLCu­VD infestation. The objectives of this study were to esti­ma­te, in different genotypes of cotton: a) WC (μg/cm ² of leaf area), b) rate of CLCuVD infestation, and c) relationship between WC and CLCuVD infestation.

Cuticle is the first layer of defense for protecting plants from environmental adversities. Cuticle consists of cutin/cutan matrix to which the cuticular waxes are either embedded (intra-cuticular waxes) or present on the outer layer of this matrix (epi-cuticular waxes) ( Yeats & Rose, 2013 ). These cuticular waxes are reported to provide protection to plants against various biotic and abiotic stresses ( Shepherd & Griffiths, 2006 ; Yeats & Rose, 2013 ; Schuster et al. , 2016 ; Xue et al. , 2017 ). In this study, a significant negative correlation was found between WC and %DI. This showed that cuticular wax had a role in confering resistance to cotton plants against CLCuVD infestation. This role may be manifested by the control of density in population of whiteflies, which are the vectors of the virus. The cotton genotypes with more WC were not favored by whiteflies for feeding and thus there was no transmission of CLCuVB to these genotypes, rendering these genotypes resistant to CLCuVD infestation. The findings of our research support a new indirect role of wax for giving tolerance against a viral disease in cotton. Previously, cuticular wax was reported to have a role for giving resistance to another insect, Hessian fly, attacks. Kosma et al. , (2010) reported that resistant plants to Hessian fly attack produced more waxes and cutin than susceptible plants. In a similar manner, cuticular wax might checked feeding of whitefly and thus inhibited transmission of CLCuVB to the body of cotton plant. Otherwise, there might be some other mechanism such as cuticular wax components’ involvement in signal transduction and activation of defense mechanism against CLCuVB. Lipids, a major constituent of cuticular wax, are reported to mediate defenses against pathogen attack ( Okazaki & Saito, 2014 ). In case of CLCuVB, this signaling role of cuticular wax will open new horizons of research endeavors. In the near future, efforts will be carried out to study the role and expression of these possible mechanisms.

In Pakistan, currently the most prevalent strain of virus, causing CLCuVD, is CLCuVB ( Hassan et al. , 2016 ). Six cotton genotypes (A-7233, B-557, A-162, BLANCO-3363, CIM-473, and SLH-2010-11), which did not show any signs of CLCuVD infestation during both 2015 and 2016, possess highest level of tolerance against CLCuVB. In this study, cotton genotypes with ≤ 10 μg/cm ² WC were found less tolerant or susceptible to CLCuVB attack. It indicated that to resist CLCuVB, a cotton genotype must possess a minimum threshold quantity of WC, otherwise it would be rendered susceptible to CLCuVB attack. On the other hand, a cotton genotype with ≥ 40 μg/cm ² WC was found tolerant to the attack of CLCuVB. It implied that a judicious quantity of cuticular wax provided required thickness of wax barrier which made hindrance in feeding of whitefly. Even though there was a uniform population of whiteflies in the experimental area, whiteflies could feed only on leaves of those cotton genotypes with less cuticular wax content and, thus, with thin layer of wax on their leaves. On the basis of these findings, it is suggested that the quantity of WC is critical for resistance to CLCuVB and may be used as a useful indicator for screen tolerance or susceptibility of cotton genotypes.