Begomovirus and DNA satellites diversity in Bemisia tabaci from cotton growing areas of Pakistan

Cotton leaf curl disease (CLCuD), a major threat to cotton production in Pakistan and northwestern India, is caused by a complex of begomoviruses transmitted by the whitefly Bemisia tabaci. These begomoviruses are typically associated with Cotton leaf curl Multan betasatellite (CLCuMuB) and often alphasatellites. Historically, the Burewala strain of Cotton leaf curl Kokhran virus (CLCuKoV-Bur) with the Burewala strain of CLCuMuB (CLCuMuBBur) was the dominant CLCuD strain in Pakistan. This study investigates the diversity and distribution of begomoviruses and their associated satellites in B. tabaci populations across major cotton-growing district of Punjab (including Faisalabad, Burewala, Vehari, and Rahim Yar Khan) and Sindh (Tando Allah Yar, Dadu, Moro, and Sakarand) of Pakistan, providing critical insights into their role in CLCuD epidemiology. The full-length begomoviruses (n = 41), betasatellites (n = 20), and alphasatellites (n = 24) were amplified, cloned and sequenced from B. tabaci (n = 56) collected during 2012-2014. Phylogenetic analyses were performed for isolates classification, and recombination events were investigated using Recombination Detection Program (RDP) and SimPlot. Phylogenetic analyses revealed a complex landscape of viral diversity, with Cotton leaf curl Kokhran virus - Burewala strain (CLCuKoV-Bur) (28/41 isolates, 68%) and its cognate betasatellite, Cotton leaf curl Multan betasatellite - Burewala strain (CLCuMuBBur) (18/20 isolates, 90%), as the dominant species.  Cotton leaf curl Multan alphasatellite (14/24 isolates, 58%) was the most prevalent satellite. The predominant B. tabaci cryptic species, Asia II 1 (or Asia–Pacific Islands–Australia, (ASIA) putative cryptic species), harbored a greater diversity of viruses and satellites compared to the less prevalent MEAM 1 (or North Africa–Middle East (NAFME) putative cryptic species). Recombination events were observed in 34% of begomovirus isolates and 40% of satellite sequences, underscoring the rapid evolutionary adaptation of these pathogens. The predominant B. tabaci cryptic species, Asia II 1 (or Asia–Pacific Islands–Australia, (ASIA) putative cryptic species), harbored a greater diversity of viruses and satellites compared to the less prevalent MEAM 1 (or North Africa–Middle East NAFME putative species). Unlike prior studies, this work is the first to identify full-length genome sequencing of begomoviruses and their associated satellites, isolated directly from B. tabaci populations, with cryptic species identification, providing a comprehensive analysis of virus-vector associations. These findings enhance our understanding of CLCuD’s molecular epidemiology and inform targeted management strategies.