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Molecular computing was used to investigate the possible causal agents of chilli crop samples showing mixed symptoms of yellow leaf curl and little leaf type diseases in the Uttar Pradesh province, India. Total genomic DNA was extracted from twenty-five samples and amplified by PCR using a universal primer pair for begomovirus and phytoplasma. Mixed infection samples show positive amplified products for begomovirus (DNA-A and betasatellite) and phytoplasma (16S rRNA and Sec A). The identified begomovirus from chilli samples was identified as a strain isolate of the previously described Chilli Leaf Curl Virus (94.2% nucleotide sequence identity), which is known to infectSolanum lycopersicon, in Oman, whereas the 16S rRNA was identified from the sourceCandidatus Phytoplasma trifolii(99.04% nucleotide sequence identity), which is known to infect Helichrysum flowering plants in India. Subsequently, molecular computing research based on phylogenetic interweaves, putative recombination, amino acid selection, and genetic diversity were investigated, revealing divergent evolutionary patterns with significant variation and recombination events. The majority of the sequence variations observed in begomovirus and phytoplasma were caused via inter- and intra-specific recombination. These findings could be the firstin silicocombined infection analysis of ChiLCV andCa.P.trifoliiin a chilli crop in India, revealing the potential adaption and evolution of begomovirus and phytoplasma to a new geographic range and crop.

During the winters of 2018 to 2020, witches’ broom, phyllody, flat stem, little leaf, yellowing and stunting symptoms were recorded in Mexican and French marigold fields in four states of India. The average recorded disease incidence ranged from 3 to 30% in different states. The ~1.2 kb product of 16S rRNA gene was amplified in symptomatic marigold samples using universal phytoplasma-specific nested primer pairs. Sequence comparison, phylogenetic and virtual RFLP analysis of 16S rRNA gene sequences identified the phytoplasmas in marigold samples as enclosed in the 16SrII-C subgroup for the strains from Delhi and in the 16SrI-B subgroup for the strains from Tripura, Uttar Pradesh and Maharashtra. Phytoplasma presence was further confirmed amplifying four genes (secA, rp, secY and tuf) in the symptomatic marigold species from all the surveyed places. Moreover, six weed species growing in marigold fields resulted positive for phytoplasmas. Phyllanthus niruri (Delhi) was tested positive for 16SrII-C subgroup phytoplasma, however, Ageratum conyzoides, Acmella calva and Ludwigia perennis (Tripura), Cannabis sativa (Uttar Pradesh) and Parthenium hysterophorus (Maharashtra) were tested positive with 16SrI-B subgroup related phytoplasma strains. Four leafhopper species were also detected positive for 16SrI-B subgroup collected from marigold fields at Tripura, Uttar Pradesh and Maharashtra and were identified as putative vectors for identified marigold phytoplasma strains. The report of association of 16SrII-C subgroup phytoplasma in Mexican marigold and P. niruri and 16SrI-B subgroup phytoplasma in L. perennis and A. calva are new reports in world.

Symptoms of little leaf were observed in brinjal plants variety VNR218 at five different locations near Gorakhpur, Deoria, Padrona, Pipraich and Kushinagar, Eastern Uttar Pradesh, India during 2018–2019. Ten symptomatic samples showing little leaf and stunted growth and three asymptomatic brinjal plants were collected during survey. Total genomic DNA was extracted from all the collected samples and primed with primers P1/P7 and R16F2n/R16R2. ~ 1280 bp amplicons were obtained from all the ten symptomatic brinjal plants. Expected size amplicons were also amplified from four BLL samples with primers specific to 16SrVI group of rp and secY genes. All the amplified products were sequenced and submitted to GenBank. On the basis of sequence comparison analysis of 16SrRNA, rp and secY gene showed 99–100% similarity with the ‘Candidatus Phytoplasma trifolii’ strain. Evolutionary and RFLP analysis also proved the association of 16SrVI-A group of phytoplasma with brinjal little leaf Gorakhpur isolate. Thus, the association of 16SrVI group and A subgroup phytoplasma with brinjal little leaf Gorakhpur isolate is the first report from Eastern Uttar Pradesh, India.

Phytoplasma suspected symptoms of little leaf and chlorosis were observed on Setaria verticillata grass at DDU Gorakhpur University Campus, Gorakhpur,Uttar Pradesh, India during 2018. To confirm the presence of phytoplasma, total DNA was extracted followed by PCR assay utilizing phytoplasma primers P1/P7 and R16F2n/R16R2. The ~ 1.2 kb amplicon was obtained by nested PCR assay from symptomatic Setaria samples. The amplified product was directly sequenced, aligned and submitted to GenBank. Comparison of 16S rRNA gene sequence of Setaria grass isolate shared 100% sequence identity with strains of 16SrII-D phytoplasma group (Ca. Phytoplasma aurantifolia). This is the first record of the occurrence of 16SrII-D subgroup phytoplasma in a weed grass, S. verticillata in India.

Cannabis sativa plants growing nearby sugarcane fields affected with grassy shoot disease at Muzaffarnagar, Uttar Pradesh, India, showed severe witches’ broom symptoms. The DNA from symptomatic Cannabis and sugarcane samples was isolated and subjected to nested PCR assays with universal phytoplasma-specific primer pairs (P1/P7 and R16F2n/R16R2). A DNA fragment of ~ 1.2 kbp was amplified in all the symptomatic samples of sugarcane and Cannabis with R16F2n/R16R2 primer pair but was absent in non-symptomatic samples. Pairwise sequence comparison, phylogeny and in silico RFLP analysis of 16S rDNA sequences confirmed the identity of a rice yellow dwarf phytoplasma group-related strain (16SrXI-B) associated with Cannabis which suggested its role as potential natural reservoir of SCGS phytoplasma in nature.

Sugarcane grassy shoot (SCGS) disease is associated with the presence of 16SrXI group phytoplasmas that are transmitted by leafhoppers; limited studies have been performed in India toward its natural transmission. To determine the insect vectors that transmit the disease in nature, leafhopper species from SCGS-infected fields at Shahjahanpur, Central Uttar Pradesh, India, were collected and analyzed for phytoplasma presence using nested polymerase chain reaction with phytoplasma-specific primers. An ~1.2-kb amplified DNA fragment was detected in nested PCR from the three major leafhopper species, viz. Maiestas portica (Melichar), Exitianus indicus (Ross) and Cofana unimaculata (Signoret), and the symptomatic sugarcane leaves of variety CoS 07250. BLASTn analysis of ~1.2-kb 16S rDNA partial sequences obtained from symptomatic sugarcane plants and these leafhoppers revealed 99–100 % sequence identities among themselves and 99 % identity with other reported strains of ‘Candidatus Phytoplasma oryzae’ (16SrXI group). Phylogenetic analysis of 16S rRNA sequences of SCGS, M. portica, C. unimaculata and E. indicus phytoplasma strains also indicated the closest phylogenetic relationship with those of ‘Ca. P. oryzae’ group. Transmission tests and population sampling study further confirmed that M. portica and C. unimaculata were vectors of the SCGS phytoplasma from diseased to healthy sugarcane plants. The identification of new vectors of SCGS phytoplasma suggested that these leafhopper species may be responsible for secondary spread of SCGS phytoplasma.

A phytoplasma has been detected in Oplismenus burmannii (Retz.) P. Beauv. and Digitaria sanguinalis (L.) Scop. plants with symptoms of chlorotic streaks and white leaf disease in India. By sequence and phylogenetic analyses of polymerase chain reaction-amplified rDNA sequences, the detected phytoplasma was identified as ‘Candidatus Phytoplasma cynodontis’, a member of the 16SrXIV group, and the causal agent of Bermuda grass white leaf disease. This is the first report on the occurrence of ‘Ca. Phytoplasma cynodontis’ in O. burmannii and D. sanguinalis grasses.

Portulaca oleracea L., an exotic weed with high medicinal value displaying typical phytoplasma associated symptoms, such as little leaf and yellowing were observed in sugarcane fields at Gorakhpur located in the state of Uttar Pradesh, India during 2013. Molecular analysis using nested-PCR with primer pairs P1/P7 and R16F2n/R16R2, evidenced that the phytoplasma found in all symptomatic plants. The nested PCR amplified product was sequenced. The approximately 1.2 kb PCR products that were observed in symptomatic Portulaca leaves had the highest nucleotide sequence similarity (99%) with members of ‘Ca. P. asteris’ group phytoplasma. Based on the BLAST analysis, the phytoplasma was classified as a member of the group 16SrI. Phylogenetic analysis also showed this phytoplasma as closely related to the reference strain for the group 16SrI. The P. oleracea phytoplasma was therefore assigned as 16SrI group and is the first report in India. Representatives of 16SrI phytoplasmas have been described in several cultivated and weed species in India and P. oleracea is an additional host species for this phytoplasma.

Little leaf symptoms were observed on Ranunculus sceleratus at Gorakhpur, Uttar Pradesh, India and collected samples resulted positive using PCR assays with phytoplasma universal primers P1/P7 followed by nested PCR with R16F2n/R16R2 primers. Selected nested PCR amplified products were cloned and sequenced. Sequence comparison revealed 99% identity with ‘Candidatus Phytoplasma cynodontis’. Phylogenetic analysis and computer-simulated RFLP patterns further confirmed that the phytoplasmas associated with little leaf disease of R. sceleratus are related to ‘Ca. P. cynodontis’ and belong to 16SrXIV-A subgroup. This is the first report of 16SrXIV-A phytoplasma association with little leaf disease of R. sceleratus in the world.

Phytoplasma was detected in Parthenium hysterophorus displaying symptoms of witches’-broom disease by direct and nested polymerase chain reaction using universal primers specific to 16S rRNA gene of phytoplasma. Sequence identities of 99% were found to several isolates of ‘Candidatus phytoplasma asteris’ (aster yellows, 16SrI group).