First Report of Okra enation leaf curl virus and Associated Cotton leaf curl Multan betasatellite and Cotton leaf curl Multan alphasatellite Infecting Cotton in Pakistan: A New Member of the Cotton Leaf Curl Disease Complex

Abstract

Cotton (Gossypium hirsutum L.) is an important and widely cultivated crop in Pakistan, upon which many rely for economic security. Cotton leaf curl disease (CLCuD) is caused by a complex comprising of more than eight species in the genus Begomovirus (family Geminiviridae) with associated betasatellite and alphasatellites. During 2011, characteristic symptoms of leaf curl disease were widespread (>40%), and the whitefly Bemisia tabaci (Genn.) vector of the leaf curl complex was abundant in commercial cotton fields in Burewala, Pakistan. Symptoms included vein thickening, upward or downward leaf curling, and foliar enations. To test for the presence of a begomovirus(es), total DNA was extracted from 100 mg of symptomatic leaf tissues from five different plants (isolates CLCuDBur1 to 5) using the CTAB method (1). Total DNA extracts were used for rolling circle amplification (RCA) using TempliPhi DNA Amplification Kit (GE Healthcare). Of the five field isolates, the RCA product for only one, CLCuDBur3, digested with HindIII, produced an apparently full-length ~2.7 kb fragment, suggesting that CLCuD-Bur3 represented a distinct isolate. The 2.7-kb fragment was cloned into the plasmid vector pGEM-3Zf+ (Promega, Madison, WI). To test for the presence of associated alphasatellites and betasatellites, the PCR primers, AlphaF/R and BetaF/R (2), were used to amplify the putative 1.4-kbp molecules. The resultant 1.4-kb PCR products were ligated into the pGEMT-Easy vector and cloned. Cloned inserts for each were subjected to DNA sequencing, bidirectionally. The cloned monopartite, helper begomovirus genome (HF567945), one betasatellite (HF567946), and one alphasatellite (HF567947) sequences were determined and found to be 2,742, 1,358, and 1,376 bases long, respectively. Pairwise sequence comparisons were carried out for each using the 10 most closely related species or strains (identified in GenBank using BLASTn) using MEGA5 software. The CLCuDBur3 genome sequence shared its highest identity (99.6%) with Okra enation leaf curl virus (OELCuV) (KC019308), so CLCuDBur3 is a variant of OELCuV, a begomovirus reported previously from Abelmoschus esculentus (L.) (okra) plants in India. The betasatellite and alphasatellite shared their highest nt identity at 96 and 98.7% with Cotton leaf curl Multan betasatellite (CLCuMB) (AM774311) and Cotton leaf curl Multan alphasatellite (CLCuMA), respectively (misnamed as CLCuBuA in GenBank) (FN658728). Additionally, the HindIII-digested RCA products were analyzed by Southern blot hybridization using a DIG-labeled DNA probe specific for the intergenic region of either Cotton leaf curl Burewala virus (CLCuBuV) or OELCuV. The OELCuV, but not the CLCuBuV, probe hybridized with HindIII digested RCA products (CLCuDBur3 genome), confirming the presence of OELCuV and the absence of CLCuBuV, the latter being the most prevalent begomovirus species infecting cotton in Pakistan. This is the first report of OELCuV infecting cotton plants in Pakistan, underscoring the discovery of yet another begomovirus member of the CLCuD complex. Further, the possible co-infection of cotton by OELCuV and other recognized species of the CLCuD complex could facilitate further diversification (potentially, through recombination) and lead to the emergence of new variants with the potential to cause damage to the cotton crop in Pakistan. References: (1) J. J. Doyle and J. L. Doyle. Focus. 12:13, 1990. (2) M. Zia-Ur-Rehman et al. Plant Dis. 97:1122, 2013.

Frequent Occurrence of Tomato Leaf Curl New Delhi Virus in Cotton Leaf Curl Disease Affected Cotton in Pakistan

“…The most surprising finding of the study presented here was the presence of the bipartite begomovirus ToLCNDV in cotton affected by CLCuD. A number of other geminiviruses have been identified in cotton including the mastrevirus Chickpea chlorotic dwarf virus [ 48 ], ToLCV [ 32 ] and Okra enation leaf curl virus [ 49 ]. However, these viruses were only identified across a limited area and in a few plants.…”

Molecular insight into cotton leaf curl geminivirus disease resistance in cultivated cotton (Gossypium hirsutum)

“…The postresistance breaking phase has witnessed a slow shift from CLCuKoV‐Bu to a situation more akin to that of the epidemic phase, with at least some of the earlier virus species/strains reappearing in cotton (Zubair et al ., 2017b). Although other begomoviruses, and even a mastrevirus (leafhopper‐transmitted geminivirus), were sporadically reported in cotton during all the phases for which information is available (Hameed et al ., ,b; Zaidi et al ., , ), the disease was always associated with CLCuMuB which is essentially required for symptom development (Zubair et al ., 2017a). A large collection of available germplasm has been screened in search of resistance against CLCuD and several lines identified as resistant to CLCuD complex have since become susceptible to CLCuD (Ahmad et al ., ).…”

An Insight into Cotton Leaf Curl Multan Betasatellite, the Most Important Component of Cotton Leaf Curl Disease Complex

“…However, unlike DNA-B of bipartite begomoviruses, where replication depends on the specific Rep of cognate DNA-A, CLCuMuB can be trans -replicated by diverse begomoviruses [ 56 ]. CLCuMuB is reported to infect cotton with Okra enation leaf curl virus [ 57 ] and ToLCNDV [ 4 ]. CLCuMuB has also been reported to infect non-cotton hosts like tomato ( Solanum lycopersicum ), bitter gourd ( Momordica spp.)…”