Genetic diversity of phytoplasma strains inducing phyllody, flat stem and witches' broom symptoms in Manilkara zapota in India.
ABSTRACT: During a survey performed in sapota orchards of India, from 2015 to 2018, symptoms of phyllody, little leaf, flat stem and witches' broom were observed in three states: Karnataka, Kerala and Tripura. The association of phytoplasmas was confirmed in all the symptomatic sapota samples by using nested PCR specific primers (P1/P7, R16F2n/R16R2 and 3Far/3Rev) with amplification of fragments of ~ 1.25 kb and ~ 1.3 kb. Association of three phytoplasma groups, aster yellows with flat stem from Tripura (Lembucherra), clover proliferation with phyllody symptoms at Karnataka (Bengaluru) and bermuda grass white leaf with flat stem and little leaf from Kerala (Thiruvananthapuram) and Tripura (Cocotilla) were confirmed by 16S rRNA gene sequence comparison analysis. Virtual RFLP analysis of 16S rRNA gene sequences using pDRAW32 further classified the sapota phytoplasma isolates into 16SrI-B, 16SrVI-D and 16SrXIV-A subgroups. This is the first report on identification of three phytoplasma groups in sapota in world.
Project description:Phytoplasma suspected symptoms of phyllody, witches' broom, leaf yellowing, stunting and little leaf were observed in Chrysanthemum morifolium, Bougainvillea glabra, Jasminum sambac and Callistephus chinensis during survey of flower nurseries and experimental ornamental fields at Delhi, Maharashtra, Tamil Nadu and Karnataka from 2014 to 2016. Pleomorphic bodies typical to phytoplasma structures were observed in the phloem sieve elements of ultrathin sections of all the four symptomatic ornamental plants (stem tissue) in transmission electron microscope. Amplification of 1.8 and 1.2 kb phytoplasma DNA products was observed in all the four test plants in PCR assays using universal primer pairs P1/P7 followed by nested primer pair R16F2n/R16R2, respectively. Pairwise sequence comparison, phylogeny and virtual RFLP analysis of 16S rDNA sequences confirmed the association of two phytoplasma subgroups (16SrI-B and 16SrII-D) in four ornamental plant species. 'Ca. P. aurantifolia' subgroup D (16SrII-D) was found associated with chrysanthemum phyllody and leaf yellowing at Delhi and Tamil Nadu, bougainvillea little leaf and yellowing at Delhi and Chinese aster phyllody at Bengaluru, Karnataka. However, jasmine little leaf and yellowing at Bengaluru, Karnataka and chrysanthemum stunting at Pune were found to be associated with 'Ca. P. asteris' subgroup B-related strains (16SrI-B). The identification of 16SrII-D subgroup phytoplasma infecting bougainvillea and 16SrI-B subgroup infecting jasmine are the new reports to the world. Besides weed species, Cannabis sativa showing witches' broom in jasmine fields at Bengaluru and Parthenium hysterophorus showing witches' broom symptoms in chrysanthemum fields at Delhi were identified to be caused by phytoplasma strains classified under subgroups 16SrI-B and 16SrII-D, respectively, by PCR assays and 16Sr DNA sequence comparison analysis. Among the three major leafhopper species identified, only Hishimonas phycitis was identified positive for 16SrI-B and 16SrII-D subgroups of phytoplasmas from chrysanthemum fields at Delhi and jasmine fields at Bengaluru, respectively. The identity of similar phytoplasma strains infecting ornamental species in leafhopper and the weed species in the present study suggested that H. phycitis and weeds may act as potential natural sources for secondary spread of the identified phytoplasma strains.
Project description:Rose balsam (<i>Impatiens balsamina</i>) is an important ornamental species grown worldwide for its attractive flowers and also having medicinal properties. Flat stem, little leaf, and phyllody symptoms were observed in <i>I. balsamina</i> nurseries in Uttar Pradesh and Tripura states of India during surveys from 2018 to 2020, with an incidence from 6 to 27%. Amplicons of ~ 1.2 kb were amplified in all the tested symptomatic samples of <i>I. balsamina</i> using universal phytoplasma primer pairs from different surveyed locations, but not from the asymptomatic plants. Pairwise sequence comparison, phylogeny, and virtual RFLP analysis of 16S rRNA gene sequences identified the phytoplasmas as 16SrI-B subgroup strain from Tripura (Lembucherra) and 16SrII-D subgroup strain from Uttar Pradesh (Gorakhpur and Faizabad). Phytoplasma presence and identity was further confirmed by amplifying <i>secA</i>, <i>rp</i>, <i>secY,</i> and <i>tuf</i> genes. This is the first report of 16SrI-B and 16SrII-D phytoplasmas detection in <i>I. balsamina</i> in the world.<h4>Supplementary information</h4>The online version contains supplementary material available at 10.1007/s13205-021-02666-2.
Project description:Brinjal little leaf (BLL) is a widespread disease of phytoplasma etiology in India that induces severe economic losses. Surveys were conducted in eight brinjal-growing states of India during July 2014 to September 2015 and eighteen BLL samples showing little leaf, phyllody and witches' broom symptoms were collected for phytoplasma identification. Presence of phytoplasmas was confirmed in all the eighteen BLL samples using polymerase chain reaction with phytoplasma-specific primer pairs (P1/P6, R16F2n/R16R2). Pair wise sequence comparison and phylogenetic relationship of 16S rRNA gene sequences of BLL phytoplasma strains confirmed that sixteen out of eighteen BLL strains belonged to clover proliferation phytoplasma (16SrVI) group and two BLL strains (GKP-A and GKP-B) from Gorakhpur, Uttar Pradesh, were classified under 16SrII group. Further virtual RFLP analysis of 16S rDNA sequences allowed finer classification of BLL strains into 16SrII-D and 16SrVI-D subgroups. BLL phytoplasma strains belonging to 16SrVI-D subgroup were found as the most widespread phytoplasma strains associated with BLL disease in India. 16SrVI-D subgroup phytoplasma association with two symptomatic weed species viz. Cannabis sativa subsp. sativa at Noida, Uttar Pradesh and Portulaca oleracea at IARI fields, New Delhi was also confirmed by nested PCR assays with similar set of phytoplasma-specific primers, pairwise 16S rDNA sequence comparison, phylogeny and virtual RFLP analysis. Out of five identified leafhopper species from BLL-infected fields at Noida, Uttar Pradesh and Delhi, only Hishimonas phycitis was identified as carrier and natural vector of 16SrVI-D subgroup of phytoplasmas by nested PCR assays, sequence comparison, phylogeny, virtual RFLP analysis and transmission assays.
Project description:Symptoms of excessive shoot proliferation were observed in the Njallani cultivar of small cardamom accompanied by stunting of stalks with fewer degenerated capsules at Nedumkandam Panchayat of Idukki district of Kerala in 2017. Five symptomatic Elettaria cardamomum shoot proliferation (ECSP) plant samples were collected and processed for DNA extraction and PCR assays utilizing universal phytoplasma 16S ribosomal-specific primers pair, P1/P7 followed by R16F2n/R16R2. Sequence comparison analysis of the R16F2n/R16R2 region of 16SrRNA gene showed 100% sequence identity with the 'Candidatus Phytoplasma australasia'- related strain. Phylogeny and virtual RFLP analyses of 16S rRNA gene sequences confirmed the association of 'Ca. P. australasia' strain subgroup D with ECSP disease. The association of 16SrII group was further established and validated by amplifying phytoplasma-specific multilocus candidate genes by utilizing specific primers of secA, secY, SAP11, and tuf genes. The multilocus gene sequence comparison analysis again confirmed the association of 'Ca. P. australasia' with the ECSP phytoplasma isolate. This is the first report of phytoplasma association with small cardamom.
Project description:In the 2019-2020 growing season, two corn fields located in İmamoğlu town (Adana Province, Turkey) were surveyed following the appearance of phytoplasma-like symptoms on maize plants. A total of 40 samples were collected and tested in first-round and nested PCR using universal primer pairs P1/P7 and R16F2n/R16R2, respectively. All maize-diseased plants reacted positively, whilst no PCR amplifications were obtained from asymptomatic plants. Blast sequence analysis of R16F2n/R16R2-primed amplicons from different maize isolates showed 99.2% to 100% of identity with the 16S rRNA gene of <i>Ligustrum witches'</i> broom phytoplasma (LiWBP). To gain additional molecular information on the 16S ribosomal RNA and 23S rRNA intergenic spacer region of LiWBP, not identified previously, the P1/P7-primed amplicons were also sequenced and analyzed. The results show that maize isolates from Turkey share 99.6% to 100% of identity among them, whereas the highest identity found (91%) was with members of groups 16SrII and 16SrXXV (peanut and tea witches' broom groups, respectively). This distant relationship between LiWBP and members of 16SrII and XXV was also confirmed by RFLP and phylogenetic analyses. This is the first finding of LiWBP on maize in nature, where it was found responsible for phyllody disease of corn plants in Turkey. The additional molecular information acquired in this study on the 16S-23S rRNA intergenic spacer region of LiWBP further corroborates its distant relationship to any other phytoplasma groups.
Project description:<i>Calendula officinalis</i> plants with phyllody symptoms (CaoP) were observed in Yazd and Ashkezar (Yazd province, Iran) during 2013-2016. Twenty-one symptomatic and four asymptomatic plants were transferred individually to the greenhouse and potted for the biological and molecular characterization of associated phytoplasma. The dodder transmission from symptomatic potted marigold plants, induced virescence, phyllody and witches' broom symptoms in periwinkle. Total DNAs extracted from symptomatic and symptomless plants and dodder-inoculated periwinkles were subjected to nested PCR assay using primer pairs amplifying phytoplasma ribosomal DNA. Expected PCR amplification was detected in all CaoP plant and dodder-inoculated periwinkle samples. RFLP analysis of the amplicons obtained in direct PCR with P1/P7 primers using <i>Rsa</i>I, <i>Alu</i>I, <i>Mse</i>I, <i>Hin</i>fI and <i>Hae</i>III restriction enzymes showed profiles identical to each other and related to phytoplasmas in all the 21 positive samples. R16mF2/R16mR2 amplicons from six CaoP plant samples were sequenced where consensus sequences had 100% of identity among each other. R16F2n/R16R2-trimmed sequences (1248 bp) of representative samples from Yazd and Ashkezar were deposited in GenBank under accession numbers KU297202 and MH065715, respectively. BLAST search and phylogenetic analysis showed that the CaoP phytoplasma had 99% homology and clusters with phytoplasmas in group 16SrII. Computer-simulated analysis using <i>i</i>PhyClassifier suggests that the CaoP RFLP 16S rRNA gene pattern was identical to 16SrII-D phytoplasmas subgroup. Phytoplasma strains (16SrII-D) were reported as alfalfa witches' broom disease agent previously in the same geographic areas, and it is possible that alfalfa plays a role in the epidemiology of CaoP disease or vice-versa.
Project description:<h4>Simple Summary</h4> Sesame (Sesamum indicum L.) is an important oilseed crop that is well known for its highly nutritious oil content. Due to its high oil content and nutritional properties, sesame is also known as the ‘queen of oilseeds’. Phytoplasma-associated diseases such as phyllody and little leaf are critical threats to sesame cultivation worldwide. Sesame phyllody is the leading biotic constraint drastically affecting sesame productivity and resulting in yield losses of up to 80% in major sesame-producing countries in the world. In this study, we explored the role of DNA methylation in phytoplasma infection. Whole Genome Bisulfite Sequencing and a Quantitative analysis of DNA methylation using real-time PCR (qAMP) revealed an alteration of methylation pattern upon phytoplasma infection. Few selected development and defense-related loci were either hypo- or hypermethylated. We hereby report the first methylome study in healthy and phytoplasma-infected sesame. Our study provides fundamental insights into the role of DNA methylation in the symptom development of phytoplasma infection in sesame plants. <h4>Abstract</h4> Phytoplasma-associated diseases such as phyllody and little leaf are critical threats to sesame cultivation worldwide. The mechanism of the dramatic conversion of flowers to leafy structures leading to yield losses and the drastic reduction in leaf size due to Phytoplasma infection remains yet to be identified. Cytosine methylation profiles of healthy and infected sesame plants studied using Whole Genome Bisulfite Sequencing (WGBS) and Quantitative analysis of DNA methylation with the real-time PCR (qAMP) technique revealed altered DNA methylation patterns upon infection. Phyllody was associated with global cytosine hypomethylation, though predominantly in the CHH (where H = A, T or C) context. Interestingly, comparable cytosine methylation levels were observed between healthy and little leaf-affected plant samples in CG, CHG and CHH contexts. Among the different genomic fractions, the highest number of differentially methylated Cytosines was found in the intergenic regions, followed by promoter, exonic and intronic regions in decreasing order. Further, most of the differentially methylated genes were hypomethylated and were mainly associated with development and defense-related processes. Loci for STOREKEEPER protein-like, a DNA-binding protein and PP2-B15, an F-Box protein, responsible for plugging sieve plates to maintain turgor pressure within the sieve tubes were found to be hypomethylated by WGBS, which was confirmed by methylation-dependent restriction digestion and qPCR. Likewise, serine/threonine-protein phosphatase-7 homolog, a positive regulator of cryptochrome signaling involved in hypocotyl and cotyledon growth and probable O-methyltransferase 3 locus were determined to be hypermethylated. Phytoplasma infection-associated global differential methylation as well as the defense and development-related loci reported here for the first time significantly elucidate the mechanism of phytoplasma-associated disease development.
Project description:Rotavirus (RV), is an etiological agent of acute infantile diarrhea in both humans and animals including poultry. Among the eight accepted species/types of RVs, RV-A is genetically and antigenically the most diverse. RV-A associated enteritis is a major problem in the weaning and post-weaning piglets. Due to high genetic variability in the antigenic regions, RV-A is thought to have high interspecies jumping probability. In this study, comparatively a large sized sample (n = 757) was screened, where the samples were collected from diarrheic porcine population of north (Uttar Pradesh), North eastern (Assam, Nagaland, Meghalaya, Tripura, Manipur, Mizoram and Arunachal Pradesh) and Southern states of India (Kerala, Karnataka and Tamil Nadu). The VP6 gene based reverse-transcription (RT)-PCR based screening of the samples for RV-A identified 42.4% (321/757) positivity, where highest identification was from Uttar Pradesh 119 (37.07%), followed by 74 (23.05%), 34 (10.6%), 31 (9.65%), 21 (6.54%), 15 (4.67%), 11 (3.43%), 8 (2.49%), 3 (0.93%), 3 (0.93%) and 2 (0.62%) from Assam, Nagaland, Meghalaya, Tripura, Kerala, Manipur, Mizoram, Arunachal Pradesh, Karnataka and Tamil Nadu, respectively. Percentage identity calculation of the VP6 gene sequences from different porcine RV-A revealed 77.1-97.3% identity within the Indian porcine RV-A strains of the current study. Phylodendrogram and percent identity based analysis of the amplified and sequenced full length VP6 gene confirmed the presence of new VP6 genotypes (I1 and I5). Although, there are reports of detection of porcine RV-A based on VP6 gene from India, no lineage/genotype based characterization is available for the target gene. Till date, only a single VP6 type (I2) has been confirmed from pig population of India. Here, the findings confirm the circulation of diverse RV-A strains in porcine population in India.
Project description:An investigation was carried out to identify and characterize the phytoplasma and viruses associated with the chickpea varieties showing severe stunting, leaf reddening, yellowing and phyllody symptoms during the summer season of 2018-2019 and 2019-2020 in eight states of India. The average disease incidence was recorded from 3 to 32% in different states. The presence of chickpea chlorotic dwarf virus (CpCDV) was confirmed in thirty-seven chickpea samples by amplification of CpCDV coat protein gene and sequence comparison analysis. No record of association of luteovirus, polerovirus and cucumovirus could be detected in any of the symptomatic chickpea samples by RT-PCR assay. <i>Brassica nigra</i>, <i>B. juncea, Lens culinaris</i>, two weeds (<i>Heteropogan contartus, Aeschynomene virginica</i>) and one leafhopper (<i>Amarasca biguttula</i>) were identified as new putative hosts for CpCDV. Association of peanut witches' broom phytoplasma was confirmed in twenty-eight chickpea samples, <i>Sesamum indicum</i>, five weeds hosts and two leafhopper species (<i>Exitianus indicus, Empoasca motti</i>) using nested PCR assays with primer pairs P1/P7 and R16F2n/R16Rn. The results of phytoplasma association in plants and leafhopper samples were further validated by using five multilocus genes (<i>sec</i>A, <i>rp, imp, tuf</i> and <i>sec</i>Y) specific primers. Sequence comparison, phylogenetic and virtual RFLP analysis of 16S rRNA gene and five multilocus genes confirmed the identity of association of 16SrII-C and 16SrII-D subgroups of phytoplasmas strain with chickpea samples collected from Andhra Pradesh (AP), Telangana, Karnataka, Madhya Pradesh, Uttar Pradesh and New Delhi. Mixed infection of phytoplasma (16SrII-D) and CpCDV was also detected in symptomatic chickpea samples from AP and Telangana. The reports of association of 16SrII-C subgroup phytoplasma in chickpea and 16SrII-D subgroup phytoplasma in <i>C. sparsiflora</i> and <i>C. roseus</i> are the new host records in world and from India, respectively.
Project description:Flower malformation represented by phyllody is a common symptom of phytoplasma infection induced by a novel family of phytoplasma effectors called phyllogens. Despite the accumulation of functional and structural phyllogen information, the molecular mechanisms of phyllody have not yet been integrated with their evolutionary aspects due to the limited data on their homologs across diverse phytoplasma lineages. Here, we developed a novel universal PCR-based approach to identify 25 phytoplasma phyllogens related to nine "Candidatus Phytoplasma" species, including four species whose phyllogens have not yet been identified. Phylogenetic analyses showed that the phyllogen family consists of four groups (phyl-A, -B, -C, and -D) and that the evolutionary relationships of phyllogens were significantly distinct from those of phytoplasmas, suggesting that phyllogens were transferred horizontally among phytoplasma strains and species. Although phyllogens belonging to the phyl-A, -C, and -D groups induced phyllody, the phyl-B group lacked the ability to induce phyllody. Comparative functional analyses of phyllogens revealed that a single amino acid polymorphism in phyl-B group phyllogens prevented interactions between phyllogens and A- and E-class MADS domain transcription factors (MTFs), resulting in the inability to degrade several MTFs and induce phyllody. Our finding of natural variation in the function of phytoplasma effectors provides new insights into molecular mechanisms underlying the aetiology of phytoplasma diseases.