Circular DNA enrichment sequencing reveals the viral/satellites genetic diversity associated with the third epidemic of cotton leaf curl disease

Abstract

Cotton leaf curl disease (CLCuD) is the most important limiting factor for cotton production in Pakistan. The CLCuD passed through two major epidemics in this region with distinct begomoviruses/satellites complexes. Since 2015 the disease has again started to appear in epidemic form, causing heavy losses to cotton crop, which we termed as the "third epidemic". We applied CIDER-seq (Circular DNA Enrichment Sequencing), a recently developed sequencing method for PCR-free virus enrichment to produce a full length read of a single circular viral genome coupled with Sanger sequencing to explore the genetic diversity of the disease complex. We identified a highly recombinant strain of Cotton leaf curl Multan virus and a recently evolved strain of Cotton leaf curl Multan betasatellite that are dominant in all major cotton growing regions in the country. Moreover, we also identified multiple species of alphasatellites with one distinct species, Mesta yellow vein mosaic alphasatellite (MeYVMA) for the first time in cotton. Relative abundance of virus and associated satellites was also determined by real-time quantitative PCR. To the best of our knowledge, this is the first study that determined the CLCuD complex associated with its third epidemic.

Keywords: begomovirus epidemics; begomoviruses; cotton leaf curl disease; geminiviruses; resistance breaking and mutations.

Figure 1:

The incidence of CLCuD in Punjab province of Pakistan. (A) The district wise incidence of CLCuD in Punjab province of Pakistan during last 3 years (values are given in percentage). (B) The pattern of CLCuD in Punjab province from 1997 to 2018 (Data of CLCuD incidence were collected by “Agriculture extension department, Govt. of Punjab, Pakistan”).

Figure 2:

(A) Map showing the spread of this distinct strain of CLCuMuV in Indian sub-continent since 2015. Map was generated online on https://static.edit.g.imapbuilder.net/ by providing the names (listed on left bottom) of locations. (B–D) Typical symptoms of CLCuD observed in major cotton growing areas in current study; upward leaf curling (B), severe downward leaf curling and stunted plant growth (C), and vein thickening (D).

Figure 3:

(A) The total number of molecules of each species versus the total number of complete sequences of each species of virus and satellites identified in the Pacbio data analysis. (B) Compared the molecules of each species with the presence and absence of conserved nonanucleotides sequence. (C) Compared the length of molecules that have conserved nonanucleotides sequence. (D) Relative titer of each species in three independent samples (S1–S3).

Figure 4:

neighbor-joining phylogenetic tree was reconstructed using the full-length genome sequences of different species of cotton begomoviruses. All the isolates of CLCuMuV identified in current study developed a novel clade in the major clade of CLCuMuV-Raj strain. We propose this novel clade associated with third epidemic of CLCuD in Indian sub-continent. Neighbor-joining method was used to reconstruct the tree using bootstrap test with 1000 replicates (percentage boopstrap value is shown on each branch). Two complete isolates of OELCV identified in current study are shown in square brackets. Species abbreviations used in this tree are following: Cotton leaf curl Khokran virus (CLCuKoV), Cotton leaf curl Alabad virus (CLCuAlV), Cotton leaf curl Bangalore virus (CLCuBanV), Cotton leaf curl Cameroon virus (CLCuCMV), Cotton leaf curl Barasat virus (CLCuBarV), Okra enation leaf curl virus (OELCV), Cotton leaf curl Gezira virus (CLCuGeV), Cotton leaf crumple virus (CLCrV), and Cotton chlorotic spot virus (CChSV).

Figure 5:

neighbor-joining phylogenetic tree of CLCuMuB constructed using the full-length genome sequences of all isolates of betasatellites so for identified from cotton species and the most identical sequences of betasatellites to the isolates of current study. Neighbor-joining method was used with bootstrap test (1000 replicates and percentage bootstrap value is shown on each branch). All isolates of current study were present in the main clade of CLCuMuB (identified from cotton in the Indian sub-continent). Isolates of CLCuMuB reported in cotton from China made a separate clade where one isolate is quite different and below the cutoff values of betasatellites to which we called CLCuMuB2 here. Other species of betasatellites so far reported in cotton are Bhendi yellow vein mosaic betasatellite (BYVMB), Chili leaf curl betasatellite (ChiLCB), and Tomato leaf curl betasatellite (ToLCB).

Figure 6:

neighbor-joining phylogenetic tree of all alphasatellites isolated in current study was reconstructed to infer their relationship with already reported isolates of alphasatellites. Bootstrap test was applied with 1000 replicates to validate the significant confidence level of each branch and its values in percentage are shown on each branch. In current study, we identified seven species of alphasatellites: Cotton leaf curl Multan alphasatellite (CLCuMuA), Tomato leaf curl Pakistan alphasatellite (ToLCPKA), Gossypium darwinii symptomless alphasatellite (GoDarSLA), Okra leaf curl alphasatellite (OkLCA), and Croton yellow vein mosaic alphasatellite (CrYVMA). Other species of alphasatellites used in this tree are Gossypium mustilinum symptomless alphasatellite (GoMuSLA), Eclipta yellow vein alphasatellite (EcYVA), Hollyhock yellow vein alphasatellite (HoYVA), and Okra enation leaf curl alphasatellite (OELCA).