Phyllody Symptoms Research Articles

Molecular characterization of chickpea chlorotic dwarf virus and peanut witches\u2019 broom phytoplasma associated with chickpea stunt disease and identification of new host crops and leafhopper vectors in India

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. Brassica nigra, B. juncea, Lens culinaris, two weeds (Heteropogan contartus, Aeschynomene virginica) and one leafhopper (Amarasca biguttula) were identified as new putative hosts for CpCDV. Association of peanut witches’ broom phytoplasma was confirmed in twenty-eight chickpea samples, Sesamum indicum, five weeds hosts and two leafhopper species (Exitianus indicus, Empoasca motti) 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 (secA, rp, imp, tuf and secY) 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 C. sparsiflora and C. roseus are the new host records in world and from India, respectively.

Occurrence of cpg islands in differentially methylated Loci in Sesamum indicum L. upon phytoplasma infection

Phytoplasmas are cell wall-less bacteria that inhabit the phloem, transmitted by sap sucking insects and associated with diseases in numerous plants including economically important taxa. Phytoplasmas are associated with the serious disease of sesame known as phyllody in nearly all growing countries. Currently, the disease is a significant threat to the production in the most important sesame-producing regions of the world. Symptoms of sesame phyllody include shoot proliferation, virescence, foliar yellowing, shortened internodes, small leaves, abnormal floral organs, generalized stunting, and phloem necrosis. Studies on changes in the levels of gene expression in response to phytoplasma infection are almost lacking in sesame. DNA methylation is a conserved epigenetic mark that regulates multiple processes, and its disruption can lead to developmental abnormalities. The CpG sites are DNA sequences where a cytosine followed by a guanine is often methylated to form 5-methylcytosines and are known to regulate gene expression through transcriptional silencing. In the present study, seven loci from sesame genome were chosen based on their role in development or defence that were found to be differentially methylated using whole genome bisulfite sequencing followed by in-silico analysis using Meth Primer online tool. All the loci were found to consist of one or more CpG islands. CpG island specific primers were designed to confirm their presence. Expected amplicons were obtained in both healthy and phyllody infected sesame plants.

Studies on transmission of phyllody from sesame to alternate host periwinkle (Vinca rosea)

Phyllody is a serious disease ofsesame worldwide and the Present investigation was carried out on transmission of this disease to periwinkle. Infected plants showed characteristic symptoms of witches' broom, stunting, leaf proliferation and bushy appearance. Under field condition, infected plants showed the most common characteristic phyllody symptoms. The pathogen phytoplasma could be successfully transmitted through grafting, leafhopper and dodder from infected sesame to healthy periwinkle plants. Among the methods, leafhopper vector (Orosius albicinctus) mediated transmission of phytoplasma was more efficient from infected sesame to periwinkle plants. Plants raised from infected periwinkle seeds did not show any phyllody symptoms.

Genetic diversity of phytoplasma strains inducing phyllody, flat stem and witches' broom symptoms in Manilkara zapota in India.

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 ~ 1.25kb and ~ 1.3kb. 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.

Detection of 16SrVI and 16SrIX phytoplasma groups in pot marigold and tickseed plants in northeastern Iran.

Pot marigold and tickseed are ornamental plants with many medicinal and cosmetic uses and for landscape, respectively. During a survey in 2018, phyllody symptoms were observed in high percentages in these plants in some regions of the Razavi Khorasan province (northeastern Iran). Total DNA was extracted from symptomatic and asymptomatic plants and polymerase chain reaction was carried on using universal phytoplasma primer pairs P1/P7 and nested primer pairs R16F2n/R16R2. The nested amplification of 1200-bp fragments confirmed the presence of phytoplasmas only in the symptomatic plants. BLAST search, phylogenetic analysis, and virtual RFLP patterns of cloned amplicons allowed to classify the pot marigold phyllody phytoplasma in the 16SrVI-A subgroup while tickseed phyllody was enclosed in the 16SrIX-I subgroup. This is the first report of the association of a 16SrVI phytoplasma with pot marigold phyllody in Iran and of the phytoplasma presence in tickseed.

Identification, expression and miRNA targeting of auxin response factor genes related to phyllody in the witches' broom disease of jujube.

Jujube witches' broom (JWB) disease, associated with the presence of phytoplasmas, induces huge crop losses in the woody perennial fruit tree Ziziphus jujuba. An imbalance in the phytohormone auxin is thought to be a key factor in the development of the witches' broom symptoms, and in the alteration of floral development into leafy structures, termed phyllody. The Auxin Response Factor (ARF) gene family controls auxin-responsive gene expression during plant growth and development. However, it remains unknown if the ARF genes are involved in the formation of leaf-like flowers. In the present study, sixteen jujube ARF genes were identified bioinformatically and annotated based on the Z. jujuba cv. Dongzao genome. The ZjARFs were homologous to 12 out of the 23 Arabidopsis ARFs and were distributed in 8 jujube chromosomes and 3 unmapped scaffolds. Phylogenetic analysis grouped the ZjARFs into three classes. Spatio-temporal expression analysis revealed that the ZjARF genes were differentially expressed among different tissues during normal development. The expression of seven ZjARF genes was significantly decreased from flower buds to flowering. JWB-infected jujube plants developed the typical phyllody symptoms and showed lower auxin accumulation during floral development. ZjARF1, ZjARF2, ZjARF3, ZjARF4 and ZjARF8 resulted differentially regulated after phytoplasma infection. ZjARF4 was down-regulated before and during floral development in phytoplasma-infected plants, but it was significantly up-regulated before flowering and down-regulated during flowering in the healthy plants. Target site analysis showed that miRNA167, miRNA529 and miRNA2950 could directly target ZjARF4. Together, the data showed that the auxin-controlled ARF4 gene is likely involved in the disruption of floral development in phytoplasma-infected jujube plants.

A new strain of ‘Candidatus phytoplasma asteris’ associated with bottlegourd phyllody disease in South India

Bottle gourd is an important cucurbitaceous vegetable crop and plants showing phyllody symptoms were observed in Bangaluru rural area of Karnataka state, India. To verify the presence of phytoplasmas total genomic DNA was extracted from symptomatic plants using a CTAB-based method and subjected to PCR assay using 16S rRNA gene specific primer pair P1/P7 followed by R16F2n/R2 nested primer pairs. The amplicons of about 1.2 kb were observed in all the symptomatic samples in nested PCR assays. The comparison of 16S rRNA gene sequence through pair wise sequence analysis and phylogeny analysis confirmed that the detected phytoplasma was closely related to those enclosed in the aster yellows group. The virtual RFLP analysis of the 16Sr DNA sequence showed restriction profiles very close to those of the 16SrI-S subgroup strain except for the MseI restriction profile, which suggested that this could be a new strain in the 16SrI group.

Molecular detection and identification of Brassica juncea phyllody and witches’ broom-associated phytoplasmas of group 16SrVI-A in Inner Mongolia, China

In September 2017, oilseed mustard (Brassica juncea) showing symptoms of phyllody and witches’-broom, indicative of phytoplasma infection, was observed in Ordos, Inner Mongolia, China. Using universal primers P1/P7, the fragment encompassing phytoplasmal 16S rRNA, 16S–23S internal transcribed spacer region and partial 5′ end of 23S rRNA genes was amplified; the ribosomal protein (rp) genes (rplV-rpsC region) and the partial elongation factor TU (tuf) gene of phytoplasmas were amplified using primer pairs rpF1C/rp(I)R1A and fTufu/rTufu, respectively. The 16S rRNA, rp and tuf phylogenies suggested that both the B. juncea phyllody and witches’-broom associated phytoplasmas are closely related to members of 16SrVI (clover proliferation) group. Based on in silico restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes, the detected phytoplasmas were classified into 16SrVI-A subgroup. The in silico RFLP pattern is consistent with the actual RFLP pattern of the 16S rRNA genes. This was the first record of 16SrVI-A phytoplasma infecting B. juncea in China, probably the first case of phytoplasma on oilseed mustard as well.

Molecular characterization of 'Candidatus Phytoplasma australasia' 16SrII subgroups associated with eggplant, cabbage, beetroot, and celery in Saudi Arabia

From 2015 to 2017 symptoms associated with phytoplasmas were observed in vegetable crop farms located in Uyun AlJiwa and Al-Dawadmi, cities located in the Al-Qassim Region and the Riyadh Province, respectively. Affected eggplants exhibited symptoms of phyllody, little leaves and witches broom, symptomatic cabbage showed multiple heads, beetroot showed reddening, stunting and leaves proliferation symptoms, while celery plants showed stunting and yellowing symptoms. To determine if the symptomatic samples were affected by phytoplasmas, PCR was performed with phytoplasma universal primers P1/P7 followed by a nested PCR with primer pair R16mF2/R16mR2, which target the 16S rRNA-encoding gene locus. PCR revealed that 20/20 symptomatic eggplants, 15/20 symptomatic cabbage, 26/30 symptomatic beetroot, and 9/11 symptomatic celery plants were infected with phytoplasma, and sequencing of the amplified genes revealed that the phytoplasmas belonged to the peanut witches’-broom group (16SrII). Sequence analysis using in silico restriction fragment length polymorphism (RFLP) and phylogenetic analysis revealed the presence of two closely related but distinct sequences from the 16SrII-D and the recently described 16SrII-X subgroups. The two sequences differed by a single base pair and both subgroups were detected in cabbage, beetroot, and celery, while eggplant was only affected by subgroup 16SrII-X. Sequence analysis of the chaperonin-60 universal target confirmed the classification of these phytoplasma strains in the 16SrII group and their close phylogenetic relationship. This is the first report of eggplant, cabbage, beetroot, and celery affected by phytoplasma in Saudi Arabia, and the first report of these plant hosts affected by the 16SrII-X subgroup.

Filodia de la margarita (Dimorphotheca sinuata) asociada a Candidatus phytoplasma asteris

En Montecillo Texcoco, Estado de México se observaron plantas de margarita (<em>Dimorphotheca</em> <em>sinuata</em>) exhibiendo síntomas de filodia, virescencia y proliferación de ramas axilares putativos a los inducidos por fitoplasmas. Por lo que el objetivo de esta investigación fue detectar el fitoplasma asociado a estos síntomas mediante PCR con los iniciadores P1/P7 y R16F2n/R2. Se detectó la presencia de fitoplasmas en tejido foliar sintomático. El producto de PCR de 1200 pb obtenido de la PCR se secuenció y sometió a digestión con las enzimas de restricción MseI (Tru91), AluI, Kpn1 y HaIII, los patrones de restricción evidenciaron que el fitoplasma presente en <em>Dimorphotheca</em> <em>sinuata</em> es <em>Candidatus</em> phytoplasma asteris (16SrI). Las secuencias obtenidas fueron depositadas en la base de datos del NCBI y tuvieron una similitud del 99% con <em>Candidatus</em> phytoplasm asteris grupo 16Sr1-B de Irán (MH638316.1). Se realizó un análisis filogenético con el método de Neighbour-Joining, en el cual el aislamiento detectado en margarita, se agrupó con <em>Candidatus</em> phytoplasma asteris. De acuerdo con los síntomas observados en campo, análisis de patrones de restricción, secuenciación y filogenia, indican que la filodia en margarita está asociada a <em>Candidatus</em> phytoplasma asteris, relacionado filogenéticamente al grupo 16Sr1-B.

Detection and identification of 16SrXIII-F and a novel 16SrXIII phytoplasma subgroups associated with strawberry phyllody in Chile

Since the year 2015, strawberry plants with symptoms of phyllody, achenes’ hypertrophy and leaf reddening have been observed in several fields in various regions of Chile. In recent surveys on five individual fields in Biobio and Araucania regions, samples were collected from symptomatic plants and DNA was extracted. Two genomic regions corresponding to the 16S rRNA and tuf genes were amplified by nested PCR using phytoplasma universal primers. Three individual clones of each amplicon from each sample were then sequenced. The RFLP analyses of the 16S rDNA amplicons showed that all of the symptomatic plants were infected by phytoplasmas enclosed in the 16SrXIII group. This result was confirmed by phylogenetic analysis with tuf amplicons. RFLP similarity coefficients indicated the presence of two phytoplasmas, one belonging to the 16SrXIII-F subgroup, while the other resulting not enclosed in any of the described subgroups and therefore it was assigned to the newly proposed 16SrXIII-K subgroup.

First report of 16SrII-D phytoplasma associated with eggplant phyllody in China

In April 2018, symptoms of phyllody, witches’ broom and little leaf were observed on eggplant on a farm located in Huiyang district, Guangdong province, China. Total DNA was extracted from the leaves of symptomatic plants and subjected to molecular detection using the phytoplasma universal primers R16mF2/mR1, P1/P7 and SecAfor1/SecArev3, which showed PCR products of the expected 1.4 kb, 1.8 kb and 0.8 kb fragments, respectively. The 16S rDNA sequence showed the highest similarity (100%) with ‘Celosia argentea’ phytoplasma strain Ca1 (GenBank accession KX426374), Crotalaria witches’ broom phytoplasma strain CrWB-Hnsy1 (GenBank accession EU650181) and pear decline phytoplasma (Taiwan II) isolate Heping (GenBank accession EF193157) from the 16SrII group. Phylogenetic analyses based on 16S rDNA and secA gene sequences confirmed the closer relationship of the eggplant phyllody phytoplasma (EPP) strain with the 16SrII-D subgroup. Virtual restriction fragment length polymorphism (RFLP) analysis of the 16S rDNA sequence indicated the eggplant phyllody phytoplasma belongs to subgroup D within the 16SrII group (16SrII-D). This is the first report of eggplant phyllody phytoplasma in China.

Crystal structure of phyllogen, a phyllody-inducing effector protein of phytoplasma

Phytoplasmas are plant pathogenic bacteria that often induce unique phyllody symptoms in which the floral organs are transformed into leaf-like structures. Recently, a novel family of bacterial effector genes, called phyllody-inducing genes (phyllogens), was identified as being involved in the induction of phyllody by degrading floral MADS-domain transcription factors (MTFs). However, the structural characteristics of phyllogens are unknown. In this study, we elucidated the crystal structure of PHYL1OY, a phyllogen of ‘Candidatus Phytoplasma asteris’ onion yellows strain, at a resolution of 2.4 Å. The structure of PHYL1 consisted of two α-helices connected by a random loop in a coiled-coil manner. In both α-helices, the distributions of hydrophobic residues were conserved among phyllogens. Amino acid insertion mutations into either α-helix resulted in the loss of phyllody-inducing activity and the ability of the phyllogen to degrade floral MTF. In contrast, the same insertion in the loop region did not affect either activity, indicating that both conserved α-helices are important for the function of phyllogens. This is the first report on the crystal structure of an effector protein of phytoplasmas.

The Harmfulness of Phoma Stem Canker, Sclerotinia Stem Rot, and Phytoplasma on Winter Oilseed Rape with Regard to Czech Breeding Programs

New genotypes of winter oilseed rape bred under the Czech breeding program were subjected to performance tests within the framework of inter-institutional pre-tests. At four monitored localities in the Czech Republic, field trials were carried out on 50 genotypes in the years 2015 to 2017. In these trials, evaluations were made on infestation by the fungal pathogens Leptosphaeria maculans, Leptosphaeria biglobosa, and Sclerotinia sclerotiorum, as well as the level of infestation caused by phytoplasmas. The evaluation of new oilseed rape genotypes confirmed a medium to high level of resistance to phoma stem canker and Sclerotinia stem rot compared to the resistant reference varieties. The most severe infestations caused by Leptosphaeria spp. were observed in Chlumec nad Cidlinou in 2015 (mean severity score 22.38%) and in Opava in 2016 (mean severity score 20.72%). The same trend was observed for infestations caused by S. sclerotiorum, where the most damage to trial plots were observed at the same localities, in 2015 in Chlumec nad Cidlinou (mean severity score 36.85%) and in 2016 in Opava (mean severity score 18.84%). PCR determination of Leptosphaeria spp. showed that 98% of sampled leaves in autumn season showing symptoms were infected by L. maculans, while the remaining 2% were identified as bearing L. biglobosa. Molecular detection of infected parts of oilseed rape plants with symptoms of phyllody confirmed in all cases the presence of phytoplasma isolates from the 16SrI Aster Yellows group.

Multilocus gene characterization of phytoplasmas in 16SrII-D subgroupassociated with Coreopsis grandiflora little leaf disease in India

In September 2018 symptoms of little leaf were observed in Coreopsis grandiflora located in flower nurseries at IARI campus, New Delhi. Little leaf and phyllody symptoms were also observed on nearby growing ornamental hedge plants belonging to Duranta genus and in a weed (Parthenium hysterophorus). Amplicons of ∼1.25 kb were consistently amplified with nested PCR assays using DNA extracted from the symptomatic Coreopsis, Duranta and Parthenimum samples, while no amplification was observed with any of the asymptomatic samples of the same species. Leafhoppers (Empoasca sp.) collected from the nurseries of Coreopsis were also found phytoplasma positive using the same nested PCR protocol. BLAST and pair wise sequence comparison of the R16F2n/R2 region of 16S rRNA gene of C. grandiflora little leaf (CGLL), Duranta sp. little leaf (DLL), P. hysterophorus phyllody (PHP) and Empoasca sp (Em) revealed 99.8% sequence identity with the ‘Candidatus Phytoplasma australasia’ sequence. Phylogeny and virtual RFLP analysis with iPhyclassifier confirmed the affiliation to ‘Ca. P. australasia’, subgroup 16SrII-D of all these phytoplasma strains. Amplicons of the expected size were also consistently amplified from these strains with primers specific to 16SrII group of SAP11, imp and tuf genes. All the amplified products were sequenced and confirmed the phytoplasma affiliation to 16SrII-D subgroup. This is the first report of 16SrII-D group phytoplasma presence in C. grandiflora showing little leaf disease.

Multigene characterization of a ‘Candidatus Phytoplasma australasia’ related strain associated with phyllody disease of coriander in India

Coriander is one of the important seed spices cultivated throughout India. The yield of the crop is very low due to the presence of many pests and diseases that are major constraints in the production. Among them phytoplasmas are one of the most economically important pathogens associated with significant yield losses. During a survey, coriander plants exhibiting phyllody symptoms were collected from Chikkabalpur dist, Karnataka (India) and the phytoplasma presence was detected through PCR using 16Sr RNA, secY, ribosomal protein (rp) and secA genes specific primers. All symptomatic coriander samples gave amplification using primers for conserved and less conserved phytoplasma genetic regions. In particular, the virtual RFLP pattern of the 16S rRNA gene, similarity coefficient 0.97 to ‘Ca. P. australasia’ enclosed it the subgroup 16SrII-D with some restriction enzyme differences. Based on the threshold similarity coefficient, this phytoplasma is related to ‘Candidatus Phytoplasma australasia’, however it shows some 16S ribosomal differences that could allow its differentiation from it. This is the first report of phytoplasmas associated with the phyllody disease of coriander from India.

Characterization of a 16SrII subgroup D phytoplasma strain associated with Calendula officinalis phyllody in Iran.

Calendula officinalis 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 RsaI, AluI, MseI, HinfI and HaeIII 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 (1248bp) 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 iPhyClassifier 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 agentpreviously in the same geographic areas, and it is possible that alfalfa plays a role in the epidemiology of CaoP disease or vice-versa.

Identification of ‘Candidatus Phytoplasma asteris’ causing sesame phyllody disease and its natural weed host in Jammu, India

During May–June 2016, little leaf and phyllody symptoms were observed on 8% of sesame crop in Chakbhalwal and Kotbhalwal regions of Jammu, India. Phytoplasma suspected witches’ broom symptoms on Cannabis sativa subsp. sativa was also observed in and around sesame fields with the incidence varied from 5 to 12% in different fields. Total genomic DNA from both symptomatic sesame and Cannabis plants were subjected to nested PCR assays using two sets of universal phytoplasma specific primers P1/P7 followed by R16F2n/R16R2 and 3Far/3Rev to amplify the 16S rDNA fragments. First round PCR amplification with primer pair P1/P7 did not yield expected 1.8 kb products of 16S rRNA gene region from any of the symptomatic test samples. However, DNA specific fragments of approximately 1.2 and 1.3 kb were amplified from sesame and C. sativa sub sp. sativa by using R16F2n/R16R2 and 3Far/3Rev primer pairs, respectively. BLASTn comparison and phylogenetic analysis of 16SrDNA phytoplasma sequences of sesame and cannabis phytoplasma strains revealed the association of ‘C. Phytoplasma asteris’ (16Sr I group) with symptomatic samples. The virtual RFLP analysis of the 16Sr DNA sequence of sesame and cannabis further identified as phytoplasma strains of 16SrI-B subgroup.

Molecular characterization of phytoplasmaassociated with four important ornamental plant species in India and identification of natural potential spread sources.

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 1.2kb 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.

Delineation of a novel subgroup 16SrXIII-J phytoplasma, a 'Candidatus Phytoplasma hispanicum'-related strain, based on computer-simulated RFLP and phylogenetic analysis.

Symptoms of fruit phyllody and slow growth, which are suggestive of phytoplasma infection, were observed in strawberry plants cultivated in commercial fields. In order to provide evidence of association of phytoplasma with affected plants, assays for detecting and identifying were performed through computer-simulated restriction fragment length polymorphism (RFLP) and phylogenetic analysis. Total DNA was extracted from symptomatic and asymptomatic samples and used as template in nested PCR primed by the primers P1/Tint followed by R16F2n/16R2. Amplified DNA fragments of 1.2 kb from the 16S rRNA gene revealed the presence of phytoplasma in all symptomatic samples. Molecular detection was confirmed by electron transmission microscopy, which evidenced pleomorphic bodies in the phloem vessels. Nucleotide sequence representative of the strawberry phytoplasma shared 97.2 to 99 % similarity with phytoplasmas currently classified as members of the distinct subgroups within the 16SrXIII group. Similarity coefficient (F) values ranged from 0.70 to 0.92, indicating that strawberry phytoplasma delineates a new strain in addition to 'Candidatus Phytoplasma hispanicum'-related strains. The evolutionary tree displayed that this strain emerges as a new branch in relation to those previously described. The novel strain, designated SFP (strawberry fruit phyllody) phytoplasma represents the new 16SrXIII-J subgroup and its sequence, denominated SFP-Br02, was deposited in the GenBank database (EU719108). These findings contribute for the knowledge of the genetic diversity existing among members of the group 16SrXIII and establishes strawberry as an additional host of representatives of this group in Brazil.