Molecular characterization of Cotton leaf curl Multan virus and DNA-satellites complex associated enation leaf curl and yellow vein mosaic disease of hollyhock


 Hollyhock is one important decorative plant grown in garden beds in different region of the the world. The ornamental plant is susceptible to many diseases caused by diverse pathogens. Among these viral pathogens can cause enormous damage to the ornamental plant. The aim of the present study was identification of begomovirus and DNA sateelites is associated with the yellow vein mosaic and enation leaf curl disease complex of hollyhock. The hollyhock plants showing the typical begomovirus-like symptoms were collected from Pusa campus, New Delhi (India). To know the status of the begomovirus, the total DNA isolated from the infected hollyhock was subjected to PCR amplification using primers specific to the begomovirus. The partial (1.2 kb) genome sequencing of ten hollyhock samples indicates the associated of begomovirus (nucleotide identities is more 95% among themselves). Therefore three representative samples (H1, H2, H3) full-length genome (DNA-A, betasatellite and alphasatellite) was amplified through RCA method. The pairwise comparision of complete genome of the begomoviruses, betasatellites and alphasatellites using Sequence Demarcation Tool (SDT) showed highest nucleotide (nt) identity of 88.0 to 92.7% (DNA-A) with Cotton leaf curl Multan virus, 92.5–96.7% with Ludwigia leaf distortion betasatellite and 90.4 to 93. 2% % with Ageratum enation alphasatellite. Further recombinantion analysis showed that the begomoviruses and DNA satellites under study was recombinants of previously reported begomoviruses and DNA sattelites. This is the first report of Cotton leaf curl Multan virus and DNA satellites associated complex disease of hollyhock in India.


Introduction
The family Geminiviridae is divided into nine generas based on genome strcture, replication, vector transmission and host range (Zerbini et al. 2017), of these the begomoviruses are major group of plant viruses affecting the different crops in worldwide and are transmitted by complex morphological indistinguishable cryptic species of white ies (De Barro et al., 2011) in a persistent circulative and nonpropagative manner. Further, based on the genome structure of the begomoviruses, were further classi ed into into Old and New world, where most of begomoviruses are belongs to the old world (OW) are monopartite with few bipartite viruses, while all New world (NW) begomoviruses are bipartite. The OW begomoviruses are more diverse as compared new world as it contain AV2 gene in OW, but it was absent in NW begomoviruses . The genomes of the OW begomoviruses are single component ( (Nawaz-ul-Rehman and Fauquet, 2009).
Most of the OW begomoviruses, additionally a classes of circular ssDNA satellites (betasatellites and alphasatellites) are associated in their genome, whereas NW and few OW begomoviruses are associated deltasastellites (Zhou, 2013;Lozano et al., 2016). The betasatellites (ssDNA) are not a true satellites and play major role in symptoms modulation in their original hosts (Venkataravanappa et al., 2011).Where as alphasatellites are ture satellites and play no role in symptom induction. Both the DNA satellites are depend on the helper virus for movement, replication and encapsidation (Briddon et al., 2006). Deltasatellites do not encode any proteins and in some cases to reduce begomovirus accumulation and symptom severity. These are still depend on helper begomovirus for replication and movement (Lozano et al., 2016).
Hollyhock (Alcea rosea L., family Malvaceae) is one important ornamental plant grown in garden beds in through out the world, including India. The ornamental hollyhock plant is originated from Asia and Europe, usually having very broad leaves, grown to increase the aesthetic value of the garden beds. Apart from its aesthetic values, the owers are used for treatments of chest complaints and decoction is used to improve blood circulation (Sharma et al. 2011). The leaves and owers are used for preventing and treating breathing disorders and digestive tract problems. Despite it many uses, the ornamental plant is a natural host for many viruses (Singh and Misra, 1971;Abdel-Salam et al. 1998, Bigarre et al. 2001Choi et al., 2003;Menzel et al. 2010;Srivastava et al. 2014;Aswathappa et al 2020;Kumar et al. 2020) are causes severe damage to hollyhock. Therefore the current study was attempted to characterize begomovirus is associated with the yellow vein mosaic and enation leaf curl disease of hollyhock in India.

Virus source plants and vector transmission
Between 2018 and 2019, the hollyhock plants exhibiting symptoms of yellow vein mosaic, enation leaf curl, and vein netting were collected from Pusa campus, New Delhi, India (Fig 1). two asyptomatic samples was also collected in the same location. During collection it was also observed presence of a huge number of white ies an undersurface of the leaves indicates the infection of begomovirus. A part of samples used for transmission of disease by white ies (Bemisia tabaci), which are reared on cotton plants under controlled conditions similar to as the transmission described by Venkataravanappa et al (2017). The adult white ies were allowed to feed on the infected hollyhock plants for an acquisition access period of 24 h. After that, the white ies (ten number on each) were transferred to ten days old seedlings of hollyhock (ten) for an inoculation access period of 24h. After that insecticide (0.05% imidocloprid) were sprayed on the inoculated plants and maintained in an insect-proof net for symptom development. As there is a probability that natural infected plants may have contaminated by multiple viruses, the arti cial transmission was repeated to check whether typical symptoms were expressed remain same or different. After repeated transmission, the symptom developemt remain same, its indicates absence of multiple infection in material. Plant tissues showing the symptoms and nonsymptomatic tissues were used for analysis. The begomovirus was successfully transmitted from naturally infected to healthy hollyhock plants (10/10 plants), which developed similar symptoms at 20-25 days post-inoculation (dpi).
DNA extraction, Polymerase chain reaction, and sequence analysis To asses the stauts of suspected begomovirus in the infected hollyhock plants, Total genomic DNA was extarcted from ten infected and two non-infected hollyhock leaf samples by using CTAB method (Doyle and Doyle 1990). The infection of begomovirus in hollyhock was screened through PCR assay using the begomovirus genome (DNA-A and DNA-B component) speci c primers (Venkataravanappa et al. 2012, Rajos et al. 1993. Based on the partial genome sequencing (1.2kb fragment) of ten hollyhock samples (sample No. H1 to H10) indicates, these samples are associated with monopartite DNA virus species, closely related to Cotton leaf curl Multan virus (Nucleotide identity more than 94%). Therefore three plant samples (H1, H2, H3), which are showing distinct symptoms were used for complete genome ampli cation using rolling circle DNA ampli cation method (Venkataravanappa et al. 2016). Further to know the subgenomic components such as betasatellite and alphasatellite associated with infected hollyhock plants. Total gDNA isolated from the hollyhock plants was subjected to PCR assay using universal primer pair speci c to betasatellite (Briddon et al. 2002) and alphasatellite (Kumar et al. 2010) as described earlier for sub-genomic components ampli cation in begomovirus infected crops so far. The RCA/PCR ampli ed products were cloned into pTZ57R ⁄T vector (Thermo Fisher Scienti c) and the con rmed clones were sequenced in both orientations.

Analysis of viral genome sequences and recombination breakpoint events
The sequence similarity of the viral geneome and DNA satellites was checked at NCBI (www.ncbi.nlm .nih.gov) using Blastn. The begomoviruses (Table S1) and betasatellites (Table S2) and alphasatellites (Table S3) are showed maximum blast score were retrieved from the GenBank. The pair wise identity score between hollyhock infecting begomviruses and selected begomviruses was calculated by using SDT version 1.2 (Muhire et al. 2014). MEGA 7 software (Kumar et al. 2018) was used to draw the phylogeny using the maximum likelihood method with 1,000 bootstrapped replications by applying Kimura 2-parameter model test. Recombination in the viral geneome and DNA satellites associated with hollyhock was carried out using RDP4 (Martin et al. 2015) with default RDP settings.

Genome organization of virus infecting hollyhock
The sequence data (DNA-A and DNA satellites) that were obtained from the three hollyhock isolates (H1, H2, H3) were assembled using different bioinformatics programs (Sea View, Bioedit and Clustal X2) and deposited in GenBank under accession number MN127817, MN127818, MN127819. The complete viral genomes of three hollyhock isolates (H1, H2, H3) were determined to be 2738 to 2750 nts long and genomic organization is analogous to other monopartite begomovirus from Old Word (OW) with potentially encoding ve conserved ORFs: two ORFs: (V2 and V1), in plus strand and four ORFs (C1, C2 C3 C4) are present in minus strand of DNA component of the DNA virus. The plus strand and minus strand of DNA A were separated by an common region (CR), which contained highly conserved nonanucleotide (TAATATTAC), sequence nicked by the rolling-circle initiator protein (Rep protein) to initiate replication. The Rep protein (encoded by C1) has all conserved domains similar other begomviruses as described by Vadivukarasi et al (2006), except the GRS motif (RFFDLISPTRSAHFHPNIQRAKS) in hollyhock infecting DNA virus is different.
The pairwise comparison complete genome of three begomovirus isolates (H1, H2, H3) infecting hollyhock with other slected begomoviruses retrieved from the NCBI database using Sequence Demarcation Tool (SDT) (Muhire et al. 2014) (Table S). The analysis showed that the DNA-A sequences of three hollyhock isolates (H1, H2, H3) showed the highest nt identity of 88.0 to 92.7 % to CLCuMuV (JN678803) infecting cotton in India. The three hollyhock isolates H1, H2, H3 were also shared < 88 % nt identity with several other begomoviruses infecting different crops in India. This result also was supported by pairwise identity scores calculated between hollyhock isolates and other begomoviruses available NCBI database by using the Sequence Demarcation Tool (SDT) (Fig. 2b). Based on genome sequence comparisons and guidelines of ICTV Study group (Adams et al. 2017 Attempts were made to detect DNA B component from infected hollyhock plants using primers speci c DNA component as decribed ealier (Venkataravanappa et al. 2012, Rajos et al. 1993) resulted in no ampli cation was observed.

Genome organization of betasatellite
Most of the begomoviruses from OW are commonly associated with DNA satellites molecule, therefore PCR was performed with primers speci c of DNA satellites (Briddon et al. 2002;Kumar et al. 2010). The resulted PCR amplicon of 1.2 to 1.3 kb in size products was ampli ed from the three hollyhock samples, indicating the association of DNA satellites with infected hollyhock plants.
The 1.3-kb size fragment of betasatellites ampli ed from the three hollyhock samples (H1β, H2β, and H3β) was cloned and sequenced. The length of complete genome sequence of three betasatellites (H1β, H2β, H3β) was determined to be ranged from 1363 to 1371bp in length and are submitted to NCBI database (under accession number MN127820, MN127821, MN127822) respectively. The sequences of three hollyhock samples (H1β, H2β, and H3β) have typical features of other betasatellites reported so far in different crops (Briddon et al. 2002;Venkataravanappa et al. 2011). The betasatellites characterized in the part of the study is belong to the family Tolecusatellitidae and showed a maximum percent nt identity of 88.1 to 93.9% among themselves and 92.5-96.7% with Ludwigia leaf distortion betasatellite (LuLDB) isolates originating from the Indian subcontinents infecting okra and hibiscus (Fig 3b). Based proposed species demarcation threshold of 91% for betasatellites (Adams et al. 2017), The betasatellites identi ed in three hollyhock samples (H1β, H2β, and H3β) is an isolate of LuLDB. This result was also supportrd by Sequence Demacration tool, the three betasatellites isolated from hollyhock plants are close related to LuLDB.
A phylogenetic analysis of complete genome sequences of three betasatellites (H1β, H2β, H3β) isolated from hollyhock with selected betasatellites revaled that three betasatellites (H1β, H2β, H3β) are closely cluster with previous isolates of LuLDB originating from the Indian subcontinents infecting okra and hibiscus for which a full-length sequence is available in the databases (Fig 3a.) Genome organization of alphasatellite associated with the disease For detection of alphasatellites, total gDNA of infected hollyhock plant was ampli ed by PCR using universal primers speci c for alphasatellites (Kumar et al. 2010). The resulted PCR amplicon of 1.2 kb in size product was ampli ed in three samples were sequenced. The sequence analysis of three alphasatellites (H1D1, H2D1, H3D1) isolated from hollyhock was determined to be1364 to 1371 bp in length and submitted to NCBI database (under accession number MN127823, MN127824, MN127825). The alphasatellites sequences have similar characterstic other alphasatellites reported so far in many crops , containing a single large ORF encoding a replication associated protein (Rep) in sense orienation ). The two alphasatellites (H1D1, H2D1) shared maximum nt identity of 90.4 to 93.2% with Ageratum enation alphasatellite (AEA) (HG518790, FR772085, HE599396) infecting cotton, okra and hollyhock (Fig 4b). Based on proposed species demarcation threshold for alphasatellite (Briddon et al. 2018), the alphasatellite identi ed here is an isolate of AEA infecting okra, hollyhock and cotton are belongs the family Alphasatellitidae, subfamily Geminialphasatellitinae and genus Colecusatellite. This results was also supported phylogeny analysis showed that two alphasatellites (H1D1, H2D1) isolated from hollyhock are more homology with several isolates AEA (HG518790 and FR772085) infecting okra and hollyhock, where as the alphasatellite (H3D1) is closely cluster with AEA (HE599396) infecting cotton respectively. This result was also supportrd by Sequence Demacration tool, the three alphasatellite isolated from hollyhock plants are close related to AEA (Fig 4a)

Discussion
Hollyhock (Alcea rosea Linn.) is an important winter ornamental plant grown in different garden beds to incease the asthetic value in many places through out India. The plant is not only ornamental but it also a good source of medicines. In the present study hollyhock plants (ten samples) showing the yellow vein mosaic, enation leaf curl and vein netting symptoms are con rmed with PCR diagnostic and viral genome sequencing, indcaites the disease was associated with CLCuMaV infecting cotton in the Indian subcontinent. CLCuMaV was rst identifed in 1967 in the Multan district of Pakistan, on scattered cottin (Gossypium hirsutum) plants (Hussain, 1975, Hussain and Mahmood, 1998, Thakur, 2002, form their it has spread rapidly other cotton-growing areas of Pakistan and Indian subcontinent. The virus (CLCuMaV) not only infecting the cotton, it will also spread to the other horticutlural crops (Mansoor et al. 2000;Mansoor et al. 2003;Hussain et al. 2004). All begomviruses associated with cotton leaf curl disease were identi ed from India are infected with cotton, okra, and tomato. The infection of cotton viruses both malvaceous and non-malvaceous hosts may be high inoculum load of cotton viruses and their strains and morphological indistinguishable white y cryptic species. Hollyhock is malvaceous plant, which is commonly grown ornamental plant in the garden as well as pots in front of houses. Due to existing of morphological indistinguishable white y cryptic species complex in India, the virus may be spreading the cotton viruses into hollyhock. Delhi is near the state of Punjab a place heavily cottongrowing area is likely white y vector, carrying the virus and transmits to hollyhock. Similarly the litratured also showed that the diverse viruses are infecting ornamental plants in worldwide (He et al. 2009;Ilyas et al. 2013), but information regarding begomovirus infections in ornamental plants was very scanty (Marwal et al. 2013b). This may be due to the study of the ornamental crops viruses are neglected or are not taken into consideration while carrying out surveys and begomovirus studies.
The betasatellites and alphastellies are found associated with OW mono and bipartite begomoviruses in many crops Stanley 2006, Rouhibakhsh andMalathi 2005;Venkataravanappa et al. 2019a, b). In the present the hollyhock samples are associated with Ludwigia leaf distortion betasatellite, which is also identi ed in begomviruses infecting different crops in India (Das et al. 2008;Roy et al. 2009;Srivastava et al. 2014). Phylogenetic analysis showed that betasatellite associated hollyhock is close clustering of the Ludwigia leaf distortion betasatellite infecting begomviruses in solanaceous crops. This clearly indicates the possible spread of the betasatellite across geographic regions among them. The study also show that betasatellites does not contain iteron sequences, therefore still it depend helper virus for its replication (Kon et al. 2007;Saunders et al. 2000;Briddon et al. 2001).
The alphasatellites (H1D1, H2D1, H3D1) associated with hollyhock were identi ed in present study were closely related to the AEA infecting okra and hollyhock (Serfraz et al. 2015) and cotton respectively (Siddiqui et al. 2016). Which are similar feature of other alphastellites identi ed in many crops Venkataravanappa et al. 2019a, b). The alphasatellites are somewhat larger than betasatellites play major role in attenuated the disease symptoms and are involved in the maintenance of low level of betasatellite accumulation in the plant (Wu and Zhou 2005). It is also showed that suppress RNAi pathway in begomovirus disease complexes (Nawaz-ul-Rehman et al. 2010).
Recombination is one of the key factor for evolution and creating genetic diversity in begomoviruses (Prasanna at al. 2010;Venkataravanappa et al. 2014). The results showed that recombination has similarly led to the formation of a distinct strain of CLCuMuV and its satellites occurring in India. The recombination analysis suggested that CLCuMuV isolates have obtained at least some of its sequences from the previously reported begomoviruses, betsatellites and alphasatelites from Indian subcontinents infecting different crops. The overall the results of the recombination and phylogenetic analysis suggested that CLCuMuV and its satellites are evolved from different monopartite begomoviruses (CLCuMuV, CLCuKoV, CLCuAIV, CLCuBaV, PaLCuV, ToLCBaV, TbCSV, BYVMV), betastellites (LuLDB), and alphasatellies(SLCuKaA, AYVIA and AEA) reported previously in India on different crops.
The present study clear indicating that the cotton leaf curl Multan virus is expanding its host range by infecting hollyhock to other malvaceous hosts is a serious threat for cultivation of many ornamental and horticulture crop plants. There is a need more comprehensive survey to identify possible spread the begomovirus infections in the country to assess their losses in crop plants. This will form the basis of our future investigations.