Begomovirus and DNA-satellites association with mosaic and leaf curl disease of Solanum nigrum and Physalis minima: the new hosts for chilli leaf curl virus.

The numerous plants of Solanum nigrum L, and Physalis minima L, well-known weeds with medicinal properties in agriculture and horticulture crops exhibiting severe mosaic, enation and leaf curl symptoms, were collected from the Varanasi and Mirzapur districts of Uttar Pradesh, India. The begomovirus infection in S. nigrum and P. minima was validated by PCR using virus-specific primers. The whole genome of the represented isolate of S. nigrum (SN1), P. minima (PM1), and beta satellite was amplified, cloned and sequenced. The SDT analysis showed that the DNA-A of PM1 and SN1 isolate showed the highest nt identity of 87.4 to 99.1%, with several chilli leaf curl virus (ChiLCuV) isolates from India and Oman, respectively. The betasatellite sequence (PM1ß) obtained from the PM1 isolate showed a very low identity of 83.1-84.5%. A demarcation threshold of 91% for betasatellite species delineation has led to identifying a new betasatellite in the PM1 sample. This unique betasatellite has been named "physalis minima leaf curl betasatellite," indicating its novelty with the plant. Whereas, betasatellite sequence (SN1ß) obtained from the SN1 sample showed 86.8-91.2% nucleotide identity with ChiLCB isolates infecting several crops in Indian subcontinents. The RDP analysis of the viral genome and betasatellite of SN1 and PM1 isolates revealed recombination in substantial portions of their genetic makeup, which appeared to have originated from pre-existing begomoviruses known to infect diverse host species. The present research also highlights the potential role of these plants as significant reservoir hosts for ChiLCuV in chili plants. Supplementary Information: The online version contains supplementary material available at 10.1007/s13337-023-00850-x.

Diversity and phylogeography of begomoviruses and DNA satellites associated with the leaf curl and mosaic disease complex of eggplant.

Eggplant is one of the important vegetable crops grown across the world, and its production is threatened by both biotic and abiotic stresses. Diseases caused by viruses are becoming major limiting factors for its successful cultivation. A survey for begomovirus-like symptoms in 72 eggplant fields located in six different Indian states revealed a prevalence of disease ranging from 5.2 to 40.2%, and the symptoms recorded were mosaic, mottling, petiole bending, yellowing, and upward curling, vein thickening, and enation of the leaves, and stunting of plants. The causal agent associated with these plants was transmitted from infected leaf samples to healthy eggplant seedlings via grafting and whiteflies (Bemisia tabaci). The presence of begomovirus was confirmed in 72 infected eggplant samples collected from the surveyed fields exhibiting leaf curl and mosaic disease by PCR using begomovirus specifc primers (DNA-A componet), which resulted in an expected amplicon of 1.2 kb. The partial genome sequence obtained from amplified 1.2 kb from all samples indicated that they are closely related begomovirus species, tomato leaf Karnataka virus (ToLCKV, two samples), tomato leaf curl Palampur virus (ToLCPalV, fifty eggplant samples), and chilli leaf curl virus (ChLCuV, twenty samples). Based on the partial genome sequence analysis, fourteen representative samples were selected for full viral genome amplification by the rolling circle DNA amplification (RCA) technique. Analyses of fourteen eggplant isolates genome sequences using the Sequence Demarcation Tool (SDT) indicated that one isolate had the maximum nucleotide (nt) identity with ToLCKV and eight isolates with ToLCPalV. Whereas, four isolates four isolates (BLC1-CH, BLC2-CH, BLC3-CH, BLC4-CH) are showing nucleotide identity of less than 91% with chilli infecting viruses begomoviruses with chilli infecting begomoviruses and as per the guidelines given by the ICTV study group for the classification of begomoviruses these isolates are considered as one novel begomovirus species, for which name, Eggplant leaf curl Chhattisgarh virus (EgLCuChV) is proposed. For DNA-B component, seven eggplant isolates had the highest nt identity with ToLCPalV infecting other crops. Further, DNA satellites sequence analysis indicated that four betasatellites identified shared maximum nucleotide identity with the tomato leaf curl betasatellite and five alphasatellites shared maximum nucleotide identity with the ageratum enation alphasatellite. Recombination and GC plot analyses indicated that the bulk of begomovirus genome and associated satellites presumably originated from of previously known mono and bipartite begomoviruses and DNA satellites. To the best of our knowledge, this is India's first report of ToLCKV and a noval virus, eggplant leaf curl Chhattisgarh virus associated with eggplant leaf curl disease.

Genome characterization and host range studies of Cucumber mosaic virus belonging to the Subgroup IB infecting chilli in India and screening of chilli genotypes for identification of resistance.

Chilli pepper is an important vegetable and spice crop grown worldwide. Chilli is susceptible to various pathogens, among them mosaic disease caused by Cucumber mosaic virus (CMV) is a major constraint for its production. Roving survey was carried out for mosaic disease assessment in chilli at 35 locations comprising five districts of south eastern Karnataka, which was later confirmed for the presence of different viruses in random samples by DAC-ELISA. Results revealed the prevalence of the disease caused by CMV up to 43.00% based on visual assessment. However, only in 64 samples out of 140 infected chilli samples showed CMV infection in DAC-ELISA and revealed the mixed infection of viruses. Mechanical sap inoculation of CMV-Ko isolate induced symptoms on chilli plants, which were similar to the symptoms observed in field. Complete genome sequence of CMV-Ko (RNA1, RNA2 and RNA3) isolate was amplified, cloned and sequenced. Sequence analysis revealed that it shared 83.7-99.1% nucleotide (nt) identity with CMV subgroup IB isolates infecting different crops in India. Recombination analysis of CMV-Ko genome showed that, RNA1 and RNA2 had recombinant origin and not RNA3. Host range studies for CMV-Ko isolate showed its potential of infecting nine host plants out of 21 used for transmission. Fifty advanced chilli lines were screened against CMV-Ko isolate and 27 immune lines to CMV were identified, which can be utilized for management of disease caused by CMV in chilli. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13337-021-00713-3.

Transmission, characterization and occurrence of recombination in Indian strain of squash leaf curl China virus associated with yellow mosaic and leaf curl disease of Summer squash.

Summer squash is one of the important vegetable crops and its production is hampered by various abiotic and biotic stresses. Of the different biotic stresses, viral infections are responsible for causing great losses to this crop. Diseases caused by begomoviruses are becoming a major constraint in the cultivation of summer squash. Samples from summer squash plants exhibiting severe yellow mosaic and leaf curl symptoms were collected from the Varanasi district of Uttar Pradesh (India) and begomovirus associated with these plants was transmitted through whiteflies (Bemisia tabaci) to healthy squash plants. The relationship between causal virus and whitefly vector was determined. The minimum acquisition access period (AAP) and inoculation feeding period (IFP) required by B. tabaci to transmit the virus was determined to be 10 min and female insects have greater efficiency in transmitting virus than male insects. The partial genome of the virus was amplified by PCR (1.2 kb), cloned and sequenced from the ten infected plant samples collected from field. Partial genome sequence analysis (1.2 kb) obtained from the ten samples revealed that they are associated with begomovirus species closely related to the Indian strain of Squash leaf curl China virus (SLCCNV). Therefore, one representative sample (Sq-1) was selected and complete genome of the virus was amplified by rolling circle amplification (RCA) method. Sequence analysis by Sequence Demarcation Tool (SDT) showed that the current isolate has maximum nucleotide (nt) identity of 93.7-98.4% and 89-98.1% with respect to DNA A DNA B, respectively with Indian strains of SLCCNV infecting cucurbits in India. Recombination analysis of genomes (DNA A and DNA B components) showed that a major part of genomes likely to be originated from already known begomoviruses (ToLCNDV, SLCCNV-CN and SLCCNV-IN) are infecting cucurbitaceous crops. Serological assays such as triple antibody sandwich-enzyme-linked immune-sorbent (TAS-ELISA) assay, dot blot immunobinding assay (DIBA), immuno-capture polymerase chain reaction (IC-PCR) were developed for the detection of SLCCNV. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-021-02821-9.

Characterization of Tomato leaf curl New Delhi virus associated with leaf curl and yellowing disease of Watermelon and development of LAMP assay for its detection.

Diseases caused by begomoviruses are becoming the major limiting factors for the production of watermelon in India. Survey for the incidence of plants showing symptoms typical to begomovirus infection was conducted in watermelon fields. The study revealed that 40% of the watermelon plants were showing the yellowing and downward curling symptoms. Twenty infected samples were collected from the different farmer's fields to know the association of begomoviruses. The PCR amplification using begomovirus-specific primers resulted in an expected 1.2 kb PCR product indicating the begomovirus association with the watermelon samples. The sequence comparison results of 1.2 kb representing partial genome revealed that all sequences obtained from watermelon samples have a nucleotide (nt) identity of more than 98% among them and are maximum homology with Tomato leaf curl New Delhi virus (ToLCNDV). One watermelon sample (WM1) was selected for complete genome amplification using RCA method (rolling-circle amplification). Amplification of DNA B and no amplification of betasatellites and alphasatellite indicated this virus as bipartite. Sequence Demarcation Tool (SDT) analysis of the DNA A component of the WM1 isolate showed the maximum nt identity of 94.6-97.9% and 85.2-95.8% with ToLCNDV infecting cucurbits. The recombinant analysis showed that the genome was likely to be derived from the recombination of already reported begomoviruses (ToLCNDV, ToLCPalV, and MYMIV) infecting diverse crops. The whitefly cryptic species predominant in the begomovirus-infected watermelon fields were identified as Asia-II-5 group. The LAMP assay developed based on coat protein gene sequence was able to detect the ToLCNDV in the infected samples. Visual detection of the LAMP-amplified products was observed with the hydroxy naphthol blue. LAMP assay was also validated with ToLCNDV infected sponge gourd, spine gourd, ivy gourd, ridge gourd, and cucumber. This is the first report of ToLCNDV association with leaf curl and yellowing disease of watermelon from India and World based on complete genome sequencing.