Detection, in silico analysis and molecular diversity of phytoplasmas from solanaceous crops in Turkey

Phytoplasma-like symptoms of leaf yellowing and calyx malformation were observed in eggplant (Solanum melongena L.), upward leaves and fruit malformation in pepper (Capsicum annuum L.), and aerial tuber formation in potato (S. tuberosum L.) during the survey performed in the late season (August to September) of 2015 and 2016 in Van province (Turkey). A total of 100 samples were tested by nested-PCR using universal primer pairs to assess the sanitary status of the solanaceous crops and to characterise the phytoplasma isolates. Among them, seven samples resulted in a 1.25 kb DNA fragment, and five (two eggplants, two peppers, and one potato) were molecularly characterised (Accession No.: KY579357, KT595210, MF564267, MF564266, and MH683601). BLAST and the virtual restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes revealed the presence of two distinct phytoplasma infections in solanaceous crops: ‘Candidatus Phytoplasma trifolii’ a member of the clover proliferation group (16SrVI) and subgroup A and ‘Candidatus P. solani’ a member of the stolbur group (16SrXII) and subgroup A. The virtual RFLP analysis and calculated coefficients of RFLP pattern similarities further revealed a remarkable genetic diversity among the ‘Candidatus P. solani’ isolates infecting pepper (similarity coefficient of 0.90) and eggplant (similarity coefficients of 0.98 and 1.00) at the same geographical area. This is the first report of the natural occurrence of ‘Candidadtus P. trifolii’ in potato from the Eastern Anatolia region, Turkey.

Detection, Identification and Molecular Characterization of a 16SrII-V subgroup Phytoplasma Associated with Nicotiana plumbaginifolia Viviani

Nicotiana plumbaginifolia Viviani or commonly known as curl-leaved tobacco is an annual herbaceous plant belonging to Solanaceae family. This plant is native to Mexico, South America, and parts of the Caribbean and has been reported to be present in Taiwan since 2006. In March 2021, N. plumbaginifolia Viviani found in Yunlin County, Taiwan was observed to have phyllody, virescence, and witches’-broom which is consistent with the disease symptoms caused by phytoplasma infection. Samples of the healthy and symptomatic plants were collected for analysis of the causal agent associated with the diseased N. plumbaginifolia Viviani. Under transmission electron microscopy, the phytoplasma-like pleomorphic bodies were found in the sieve tubes of the diseased plants. The 16S rRNA-based phylogenetic analysis and the iPhyClassifier-based virtual RFLP study demonstrated that the phytoplasma identified in this study can be classified into the 16SrII-V subgroup, which is similar to the peanut witches’-broom phytoplasma, a ‘Candidatus phytoplasma aurantifolia’-related strain. Further identification of SAP54/PHYL1 and SAP11 homologues in the phytoplasma explain the disease symptoms of phyllody, virescence, and witches’-broom observed in diseased N. plumbaginifolia Viviani. The discovery of new phytoplasma plant hosts has gained scientific importance in light of the attempt to unravel an efficient strategy to fight the rapid spread of this disease which poses threat to the agricultural sector and food security in Taiwan.

New genetically distinct phytoplasmas and insect carriers associated with pine tree disease revealed by a survey in Curonian Spit, Lithuania

Our previous studies reported that phytoplasma was the causative agent of the pine disease in Curonian spit, Lithuania. In this study, insects from diseased pine trees and their adjacent areas were collected from 2016 to 2019 to further identify potential insect vectors that spread phytoplasmas. A total of 1018 phloem-feeding insects (order Hemiptera) were identified, 98.62% of which were aphids (Aphididae), and no known phytoplasma vectors were found. Results from semi-nested PCR using phytoplasma-universal primers revealed that phytoplasmas were detected in scots pine aphids (Cinara pini), waxy grey pine needle aphids (Cinara pineti), and species-unknown aphids. Further sequence analysis and virtual RFLP analysis of aphid-harbored phytoplasma strains indicated that they were closely related to ‘Candidatus Phytoplasma pini’ (16SrXXI-A), but mainly 16SrXXI-A variants, which were also main strains identified in diseased pine trees. In addition, three new phytoplasma subgroups were delineated in the present study. Subgroups 16SrXXI-C and 16SrXXI-D were unveiled from previously identified (but classification was overlooked) Lithuanian pine phytoplasma strains. Subgroup 16SrXXI-E was discovered from the newly identified aphid-harbored phytoplasmas. Further transmission trial study on these aphids will provide insights into the epidemiology, and pathosystem of pine phytoplasma diseases, as well as the disease management.

Identification of 16SrII-V Phytoplasma Associated with Mungbean Phyllody Disease in Taiwan

Mungbean (Vigna radiata (L.) R. Wilczek), an important legume crop in Asia, is primarily cultivated in the central-southern region of western Taiwan. In 2020, mungbean exhibiting typical phytoplasma-induced disease symptoms, such as witches’ broom, phyllody, virescence, and proliferation, was observed in Yunlin County, Taiwan. Moreover, the seeds harvested from diseased plants displayed premature germination. Transmission electron microscopy examination of leaf veins prepared from symptomatic mungbeans demonstrated that the occlusion of sieve tubes resulted from the accumulation of phytoplasma-like bodies in sieve elements along with filament-like structures in sieve pores. The association of phytoplasma in symptomatic mungbean was confirmed by PCR analyses of the 16S rRNA and immunodominant membrane protein genes. Further analyses of the 16S rRNA-based phylogenetic tree and the iPhyClassifier-based virtual RFLP study demonstrated that the phytoplasma-associated mungbean phyllody disease identified in this study belongs to the 16SrII-V subgroup. BLAST analysis and the phylogenetic analysis indicated that the SAP11-like protein identified in mungbean phyllody disease is identical to PnWB phytoplasma SAP11, which explains the witches’ broom phenotype observed in symptomatic mungbean. The results described in this report confirm that the 16SrII-V phytoplasma, a widely distributed phytoplasma associated with peanut witches’ broom disease in Taiwan, has also infected mungbean. This is not only the first instance of mungbean phyllody disease found in Taiwan, but also the first instance of mungbean phyllody disease causing by 16SrII-V subgroup phytoplasma.

Multilocus gene analysis reveals the presence of two phytoplasma groups in Impatiens balsamina showing flat stem and phyllody

Rose balsam (Impatiens balsamina) 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. balsamina 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. balsamina 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 secA, rp, secY, and tuf genes. This is the first report of 16SrI-B and 16SrII-D phytoplasmas detection in I. balsamina in the world.

Molecular characterization of chickpea chlorotic dwarf virus and peanut witches’ 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.

First report on clover proliferation phytoplasma related strain associated with Matthiola incana floral virescence in Uttar Pradesh, India

Matthiola incana R. Br. (Fam: Brassicaceae) is an ornamental, commonly known as hoary stock has an extremely fragrant flowers, which blooms in dense clusters in a large variety of colors. During a survey of flower nurseries in March 2019 at Indian Institute of Sugarcane Research campus, Lucknow, floral virescence (MiV) symptoms (Fig. 1 A, B) were observed in M. incana pots with an incidence of over 40%. Leaf yellows symptoms were also observed on a weed Acalypha indica (AiLY) in Matthiola nursery (Fig. 1 C). Nested PCR assays were carried out to detect and identify the possible association of phytoplasmas with MiV and AiLY symptoms. Three each of symptomatic MiV and AiLY samples and two non-symptomatic samples were collected and processed for DNA extraction from the leaf midrib by CTAB method. Hishimonus phycitis (HP) (Hemiptera: Cicadellidae) leafhopper feeding on MiV symptomatic plants was also collected and DNA was extracted. The DNA of 8 symptomatic and 4 non-symptomatic plants and from the 10 leafhopper was used as a template for PCR assays. Phytoplasma specific 16Sr RNA gene specific primers (P1/P7 and 3Far/3Rev; Schneider et al. 1995; Manimekalai et al. 2010) and multilocus genes’ specific primer pairs for secA (SecAfor1/SecArev3;SecAfo5r/SecARev2; Bekele et al. 2011), secY (SecYF1(VI)/SecYR1(VI);SecYF2(VI)/SecYR1(VI); Lee et al. 2010) and rp genes (rpFIC/rp(I)R1A; rp(VI)F2/ rp(VI)R2; Martini et al. 2007) were employed as previously described. Amplified products of ~1.3kb, ~600bp, ~1.7kb and ~1.0kb of 16S rRNA, secA, secY and rp genes of phytoplasma were consistently amplified in all the MiV and AiLY samples and in the HP leafhopper. No amplifications were achieved in any of the asymptomatic plant samples. Amplified products of all the four genes of MiV, AiLY and HP isolates were purified, sequenced and submitted in GenBank. Sequence comparison and phylogeny analysis of the sequences of the four genes of MiV, AiLY and HP isolates revealed 99% - 100% sequence identity and clustering with clover proliferation phytoplasma related strains (16SrVI group)(Fig.2 A,B,C and D). The virtual RFLP analysis of 17 restriction endonucleases corresponding to the 16S rDNA sequence of MiV, AiLY and HP phytoplasma strains by pDraw program, assigned them into a novel phytoplasma subgroup strain under 16SrVI group, since its HpaII restriction profile was different to earlier classified 16SrVI subgroups but was very close to16SrVI-E subgroup (GenBank acc. no. AY270156) (Fig 3). Earlier, peanut witches’ broom (16SrII-A) phytoplasma was identified associated with M. incana from Italy (Davino et al. 2007). However, the association of clover proliferation phytoplasma (16SrVI) related strain associated with virescence symptom of M. incana is the first report in world. The weed (A. indica) and HP leafhopper were also reported as additional hosts of 16SrVI subgroup related new strain in India, which needs further investigation. The report of a new host and new subgroup of clover proliferation phytoplasma related strain in India is having an epidemiological significance and warrants attention.

Identification of phytoplasma strains associated with witches’ broom and yellowing in Ziziphus jujube nurseries in Iran

Phytoplasma symptoms, including proliferation, witches’ broom, leaf rolling and yellowing, were observed in jujube (Ziziphus jujube) nurseries in the East of Iran. Total nucleic acid was extracted from symptomatic and symptomless plants, and was tested for phytoplasma presence using nested PCR. Amplicons of about 1.8 kb (primer pair P1/P7) and 1.25 kb (R16F2n/R16R2) were obtained from all symptomatic plants but not from symptomless plants. Restriction fragment length polymorphism (RFLP) analysis of R16F2n/R2 amplicons using KpnI, HaeIII, RsaI, AluI, HpaII, HhaI, TaqI, MseI, BfaI and ThaI restriction enzymes showed two RFLP patterns referable to 16SrI and 16SrVI phytoplasma groups. The consensus sequences of Z. jujube yellowing and witches’ broom of six samples correspond to ‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma trifolii’-related strains. Two R16F2n/R16R2 16S rDNA sequences representative of each RFLP profile, one each from witches’ broom (accession number MK379605) and yellowing (MK379604) host symptoms, were submitted to the GenBank. Phylogenetic analysis confirmed that the phytoplasma strains associated with jujube yellowing clustered within the 16SrI phytoplasma clade, and those associated with witches’ broom clustered within the 16SrVI clade. Restriction analysis confirmed that virtual RFLP patterns of the jujube yellowing and witches’ broom phytoplasma strains were identical to the reference pattern of 16SrI-B and 16SrVI-A. This is the first report of these phytoplasma strains associations with witches’ broom and yellowing in jujube plants.

Molecular identification of phytoplasmas associated with some weeds in West Azarbaijan province, Iran

During field surveys in 2013 and 2014, about 14 weed plants showing phytoplasma diseases symptoms including yellowing and witches’broom were collected and tested by polymerase chain reaction (PCR) using universal primers for 16SrRNA starting by primer pairs P1/P7 in first round PCR followed by primer pair R16F2n/R16R2 in nested PCR. The detected phytoplasmas were characterized and differentiated through sequence analysis of PCR-amplified rDNA and virtual restriction fragment length polymorphism (RFLP). The phytoplasmas detected in symptomatic horseweed<strong> </strong>(<em>Erigeron canadensis</em> L.), common madder (<em>Rubia tinctorum</em> L.), Johnson grass (<em>Sorghum halepense</em> [L.] Pers.) and Sophora root (<em>Sophora alopecuroides</em> L.) were identified as members of the clover proliferation group (16SrVI group) by construction of phylogenetic trees. Further analysis by virtual RFLP classified the phytoplasmas of <em>Erigeron canadensis</em> L. and <em>Sorghum halepense</em> L. in subgroup 16SrVI-A and phytoplasmas of <em>Rubia tinctorum</em> L. and <em>Sophora alpecuriodes</em> L. in subgroup 16SrVI-D. This is the first report on the occurrence of phytoplasma diseases of weeds in west Azarbaijan, Iran.