Association Of Phytoplasma Research Articles

Association of Phytoplasmas with a New Disease of Potato Crops in Cundinamarca, Colombia

Association of Phytoplasmas with a New Disease of Potato Crops in Cundinamarca, Colombia

First report of 'Candidatus Phytoplasma asteris' associated with witches'-broom disease of Macadamia ternifolia in China.

Macadamia nut (Macadamia ternifolia) was first introduced into China from Australia in 1910, and the main cultivation areas were Yunnan and Guangxi. It can be used as both a fruit and a therapeutic drug, with high economic value. In March 2021, it was observed that the M. ternifolia was showing witches'-broom, leaf yellowing and plexus bud in Dehong Prefecture, Yunnan Province, China. The terminal buds of infected plants were inhibited and the lateral buds are stimulated to germinate into twigs in advance. It was named the M. ternifolia witches'-broom disease, and was found in urban and rural areas of Mangshi, Lianghe, Yingjiang, Mangdong, Longchuan and Longling cities and counties. More than 40% of the plants were infected on the seven areas surveyed. The lateral stems from symptomatic and asymptomatic plants were cut to small pieces. The tissues were treated by fixation, dehydration and spraying-gold. And the tissues were observed under a scanning electron microscope (Hitachi S-3000N) (Pathan et al. 2010). The nearly spherical bodies were found in the phloem sieve cells of symptomatic plants. Symptomatic and asymptomatic plants were collected from seven areas, ddH2O was used as the negative control, and Dodonaea viscose witches'-broom disease plants were used as the positive control. The total plants' DNA extraction was conducted from 0.1 g tissue using the CTAB method (Porebski et al. 1997), and were stored at -20 °C in a refrigerator in the Key Laboratory of Forest Disaster Warning and Control at the Southwest Forestry University. The nested PCR was employed to amplify the 16S rRNA gene with the primers P1/P7 and R16F2/R16R2 (Lee et al. 1993; Schneider et al. 1993). PCR amplicon of 1.8 kb and 1.2 kb were amplified (GenBank accessions MW892818, MW892819, MW892820, MW892821). The direct PCR with primer pairs rp(I)F/ rp(I)R (Lee et al. 2003) specific to the ribosomal protein (rp) gene yielded amplicons of approximately 1.2 kb (GenBank accessions MZ442600, MZ442601, MZ442602, MZ442603). The fragment from 21 samples was consistent with the positive control, confirming the association of phytoplasma with the disease. Interestingly, the phytoplasma/span>16S rRNA gene and rp gene was also amplified from the 4 samples of asymptomatic plant, we speculated that the latent infection and hidden symptoms existed in Macadamia nut (Moslemkhani and Sadeghi 2011). A BLAST analysis of the 16S rRNA sequences of MTWB phytoplasma showed that it has a 99% similarity with Trema laevigata witches'-broom phytoplasma (GenBank accession MG755412). The rp sequence shared 99% identity with 'Salix tetradenia' witches'-broom phytoplasma (GenBank accession KC117314). An analysis with iPhyClassifier showed that the virtual RFLP pattern derived from the query 16S rDNA fragment of MTWB phytoplasma is most similar to the reference pattern of the 16Sr group I, subgroup B (OY-M, GenBank accession AP006628), with a pattern similarity coefficient of 0.99. The phytoplasma is identified as 'Ca. Phytoplasma asteris'-related strain belonging to sub-group 16SrI-B. The phylogenetic tree was constructed based on 16S rRNA gene and rp gene sequences by using MEGA version 6.0 (Tamura et al. 2013) with neighbor-joining (NJ) method and bootstrap support was estimated with 1000 replicates. The result indicated that the MTWB phytoplasma formed a subclade in 16SrI-B and rpI-B respectively. In 2013, Macadamia nut showed leaf hardness phyllody and shoot proliferation caused by 'Ca. Phytoplasma asteris' in Artemisa, Cuba. The concern is that, the macadamia phytoplasma is closely related to the subgroup 16SrI-F, and it is significantly different from the Chinese strains (Pérez-López et al. 2013). In addition, the MTWB phytoplasma was graft-transmitted from infected to healthy plants in nursery conditions (Akhtar et al. 2009; Ikten et al. 2014). And the grafted plants were positive for the phytoplasma in the nested PCR assays. It is noteworthy that the plants were seriously damaged by aphid and it was speculated that the insects of Homoptera caused the spread of the disease by sucking plant sap, thus the aphids that transmits MTWB in China must be determined to control the M. ternifolia witches'-broom disease. To the best of our knowledge, Macadamia nut is a new host plant of 'Ca. Phytoplasma asteris' in China. The newly emerged disease is a threat to Macadamia nut.

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 seed harvested from diseased plants displayed premature germination. Transmission electron microscopy examination of leaf veins prepared from symptomatic mungbean 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 ribosomal RNA (rRNA) and immunodominant membrane protein genes. Further analyses of the 16S rRNA-based phylogenetic tree and the iPhyClassifier-based virtual restriction fragment length polymorphism 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 peanut witches' broom 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 caused by 16SrII-V subgroup phytoplasma.

Association of a phytoplasma from ribosomal subgroup 16SrXII‐A with walnut witches’‐broom in Iran

Walnut trees displaying symptoms similar to those caused by phytoplasmas, including foliar chlorosis, yellowing and shortening of internodes, were found in a fruit orchard in the north of Fars province, Iran in 2020 (Fig. 1). Samples were collected and analysed by PCR to detect and identify possible phytoplasma(s) associated with diseased plants. Four diseased and four asymptomatic walnut trees were sampled and analysed for the presence of phytoplasma. Total DNA was extracted from 0.1 g of midribs using the CTAB method. Nested PCR was performed using universal phytoplasma primers P1/P7 (Deng & Hiruki, 1991) in the first round, and R16F2n/R16R2 (Smart et al., 1996) in the second. PCR products of the expected size, c. 1.2 kb, were obtained from all diseased plants but not from the symptomless ones. The nucleotide sequence was determined directly and submitted to GenBank (Accession No. MZ005313). A BLAST search showed that the phytoplasma associated with walnut witches’-broom had the highest sequence identity (99.9%) with Iranian alfalfa witches’-broom phytoplasma (KT763373), a member of the ribosomal subgroup 16SrXII-A (‘Candidatus Phytoplasma solani’). Virtual RFLP patterns obtained with iPhyclassifier (https://plantpathology.ba.ars.usda.gov/cgibin/resource/iphyclassifier.cgi) supported the sequence analysis results. Walnut witches’-broom phytoplasma was first reported in Iowa, USA and was associated with the group 16SrIII (‘Ca. P. pruni’) (Yun & Harrington 2011). In Iran, the 16SrXII phytoplasma group has been reported in alfalfa, almond, aubergine, grapevine, peach, plum and tomato (Siampour et al., 2019). It has also been identified from Cannabis sativa (Sichani et al., 2011) and Salix babylonica (Zamharir & Taheri, 2017). In Fars province, walnut orchards are adjacent to stone fruit orchards and vineyards, so it is likely that this phytoplasma has been transferred from such crops to walnut trees. Since large areas of walnut are grown in Iran, this phytoplasma may be a serious threat to walnut cultivation. To the best of our knowledge, this is the first report of the association of a 16SrXII-A related phytoplasma with walnut witches’-broom globally. The authors would like to thank Dr. Sadegh Sadeghi and Mr. Mohsen Amiri for great help in the field.

First Report of 16SrII-V Phytoplasma Associated with Green Manure Soybean (Glycine max L.) in Taiwan.

QING PI DOU, a local variety of soybean (Glycine max (L.) Merrill) with small seed size, is primarily cultivated in the southern region of Taiwan. Due to the advantage of high germination rate, fast growth and high nitrogen fixation capacity, QING PI DOU has widely used as green manure in rotation with rice to increase soil fertility in Taiwan. In the summer of 2020, phytoplasma-induced disease symptoms were observed in QING PI DOU with 23% (18/78) disease incidence in Yunlin County, Taiwan. These plants exhibited severe disease symptoms such as little leaf, yellowing, phyllody, virescence, and witches' broom compared to healthy plants. Leaf samples of the symptomatic plants were subsequently collected and examined through transmission electron microscopy (TEM), PCR, and western blotting analyses. The ultrathin sections of the diseased QING PI DOU were double-stained with uranyl acetate and lead citrate. The typical phytoplasma-like pleomorphic bodies were observed in sieve elements of leaf veins by TEM. To investigate the association of phytoplasma with the diseased QING PI DOU, total DNA extracted by the Plant Genomic DNA Purification Kit (DP022, Genemark, Taiwan) was examined by nested PCR using the phytoplasma universal primer pair P1/P7 followed by R16F2n/R16R2 (Lee et al. 1993). The 1.2 kb PCR product specific for 16S ribosomal RNA (16S rRNA) gene was only amplified from symptomatic plants but not from healthy plants. BLAST analysis demonstrated that the sequence (accession no. MW393690) of amplified DNA fragment of 16S rRNA is identical to that of GenBank accession no. NZ_AMWZ01000008 (complement [31109 to 32640]) of peanut witches' broom (PnWB) phytoplasma, a 'Candidatus phytoplasma aurantifolia'-related strain (Firrao et al. 2004). Further analysis on the virtual RFLP pattern of MW393690 generated by iPhyClassifier confirmed that the phytoplasma identified in the diseased QING PI DOU can be classified into the 16SrII-V subgroup. Samples examined by nested PCR were further selected for total cell extracts preparation and characterized by western blotting using the polyclonal antibody raised against the immunodominant membrane protein (Imp) of PnWB phytoplasma (Chien et al. 2020). An expected signal of 19 kDa specific for Imp was only detected in symptomatic plants but not in healthy plants. Moreover, the PCR products encoding SAP11 and phyllogen, the virulence factors responsible for phytoplasma-induced witches' broom and phyllody symptoms (Namba 2019), were also amplified from symptomatic QING PI DOU by PCR using the primer pairs 5'-ATGGCTCCCGAAAAAAATGATAAAGG-3'/5'-TTTTTTAGAATCATCAGGCTTTTTAG-3' (0.28 kb) and 5'-ATGGATCCAAAACTTCCAGAAACT-3'/5'-GTTTTTTTCATCATTTAAATCAT-3' (0.27 kb), respectively. Further analysis by BLAST revealed that SAP11 and phyllogen identified in symptomatic QING PI DOU are identical with those of PnWB phytoplasma. To the best of our knowledge, this report is the first to describe phytoplasma-associated soybean (Glycine max L.) witches' broom disease in green manure soybean in Taiwan.

Molecular identification of 16SrXI-B subgroup of phytoplasma related strain with yellow leaf disease of sugarcane (Saccharum officinarum) in India

In India, yellow leaf disease (YLD) is known to be caused by Sugarcane yellow leaf virus (SCYLV) and Sugarcane yellows phytoplasma (SCYP; 16SrI-B). Therefore, present study was conducted to confirm the association of specific phytoplasma with YLD in Co 0238 and CoS 510 sugarcane genotypes during 2019-2020 at Indian Institute of Sugarcane Research, Lucknow. The YLD affected genotypes were characterized by yellowing of leaf mid-ribs and leaf-lamina, leaf-tip necrosis, bunching of leaves at crown region and drying of plants with an incidence ranged from 28.26-35.81%. YLD symptomatic leaf samples were collected from the two genotypes and subjected to genomic DNA extraction by DNeasy Plant Mini Kit followed by nested PCR assay using universal phytoplasma primers (16S rDNA region). The nested PCR assays of samples from both the genotypes showed specific amplification. 16S rRNA gene sequence analyses of CoS 510 isolate (MN913611) revealed 100% identity with Sugarcane white leaf phytoplasma (SCWLP) belongs to 16SrXI-B subgroup. Similarly, virtual RFLP analyses revealed 1.00 similarity coefficient with 16SrXI-B subgroup. Thus, present study first time reports the association of SCWLP related strain with YLD of sugarcane in India. Phytoplasma infecting sugarcane are more diverse and needs systematic studies on their single or mixed infections in different varieties in India.

Characterization of ‘Candidatus Phytoplasma asteris’-related strain association with leaf yellowing of Wrightia antidysenterica (Arctic Snow) in Tripura, India

Wrightia antidysenterica is a small compact and evergreen ornamental shrub. Little leaf and yellowing symptoms were recorded on W. antidysenterica plants in June 2018 at College of Agriculture campus, Lembucherra, Tripura. The expected amplicons of ~1.25 kb and ~480 bp were obtained in symptomatic leaf samples with the phytoplasma specific universal 16S rRNA and secA gene primers. Sequence comparison analysis of 16S rRNA and secA gene confirmed the association of ‘Ca. P. asteris’-related strain. However, comparing the restriction profiles of 16S rRNA gene in virtual RFLP analysis revealed that the phytoplasma was a 16SrI group related strain. To our knowledge, this is the first report of phytoplasma association with W. antidysenterica little leaf and yellowing in world.

Association of a 16SrIX-A phytoplasma with Conocarpus erectus showing stem fasciation and its vector in Iran

Association of a 16SrIX-A phytoplasma with Conocarpus erectus showing stem fasciation and its vector in Iran

Olive tree represents a new host of a subgroup 16SrVII-B phytoplasma associated with witches' broom disease in Brazil.

Olive trees exhibiting slow development, yellowing, and high intensity of shoot proliferation with small leaves were observed in commercial plantings, in the municipality of Extrema, Minas Gerais (MG) state in 2015. The incidence of symptomatic plants was about 70% and diseased trees presented yield reduction. Here we report the association of symptomatic olive trees with a phytoplasma and describe its molecular identification. Symptomatic plants (38 trees) were sampled in three growing areas located in the same municipality. The samples consisted of bunch of leaves and young shoots. The total DNA was extracted using DNeasy® Plant Mini Kit (Qiagen, Hilden, Germany). Phytoplasma detection was conducted by nested PCR with primers P1/16S-SR (Lee et al. 2004) followed by R16F2n/R16R2 (Gundersen and Lee 1996). PCR assays generated amplicons (~1.2 kb) from 28 trees out of 38 symptomatic plants, confirming the association of phytoplasma with diseased plants. The disease was named olive witches' broom. The genomic fragments amplified by nested PCR were cloned into Escherichia coli DH5α and sequenced. The sequence representative of the olive phytoplasma was designated OWB-Br01 (Olive Wiches' Broom-Brazil 01) and deposited in GenBank under accession number MH141985. This sequence shared 99% sequence identity with phytoplasmas affiliated with 16SrVII group. According to the iPhyClassifier online tool (Zhao et al. 2009) the olive witches'-broom phytoplasma was classified as a variant of subgroup 16SrVII-B with a pattern similarity coefficient of 0.99. The phylogenetic tree showed that OWB-Br01 phytoplasma emerges from the same branch of the reference phytoplasma of the 16SrVII-B subgroup (Erigeron witches᾽-broom phytoplasma - GenBank AY034608), indicating that the olive tree phytoplasma is a member of the 16SrVII-B subgroup. The pathogenicity test was performed with 28 healthy plants (cultivar Arbequina) grown in pots, which were grafted by simple english forklift with scions obtained from olive plants (Arbequina) six years old, naturally infected by the phytoplasma. The initial symptoms were observed four months after grafting and at eight months 22 grafted plants exhibited slow growth, yellowing, and small leaves as those naturally observed in the fields. Molecular characterization allowed identify the phytoplasma as a member of the 16SrVII-B subgroup. In Brazil, representatives of the 16SrVII group were previously reported in association with diverse botanical species. Thus, a strain of 16SrVII-C subgroup was identified in sunn hemp (Flôres et al. 2013); the reference phytoplasma of 16SrVII-D subgroup was found in erigeron plants (Flôres et al. 2015); and the representative of 16SrVII-F was detected in the wild species Vernonia brasiliana. (Fugita et al. 2017). Specifically regarding subgroup 16SrVII-B, the reference phytoplasma of this subgroup was described from erigeron and periwinkle (Barros et al. 2002), while other members of this subgroup were reported in cauliflower (Pereira et al. 2016a) and ming aralia (Pereira et al. 2016b). The disease here studied is a threat since olive planting is in large expansion in Brazil. A potential control option could be use of propagative material from sources free of the pathogen. Based on our findings, olive tree represents a new host for subgroup 16SrVII-B phytoplasma, which is different from 16Sr groups previously reported as associated with olive witches' broom in other countries.

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.

Multilocus sequence analysis of Candidatus Phytoplasma aurantifolia associated with phyllody disease of gerbera from India.

Gerbera is the most popular cut flower known for its variety of colors and is grown across the world. Its production is challenged by numerous diseases affecting production and quality. During our survey, ten samples from the gerbera plants exhibiting phyllody disease symptoms were collected from Bangalore Rural District, Karnataka, India. The association of phytoplasma with the gerbera phyllody samples was confirmed by PCR using 16SrRNA, SecY, Ribosomal protein (rp) and SecA gene-specific primers. PCR products were amplified from all ten gerbera plants using phytoplasma-specific primers. The amplified PCR products were cloned and sequenced; the sequences of the ten clones were identical. Therefore, representative isolate (GePP1, Gerbera phyllody phytoplasma) was selected for further analysis. The sequence analysis showed that GePP1 shared maximum nucleotide (nt) identity of 97.1% (16SrRNA) with Eggplant big bud, 98.7 to 98.8% (SecY gene) with Tomato big bud, 99.2 to 99.6% (rp gene) with Alfalfa witches-broom (EF193371) and 99.1% (SecA gene) with Sesame phyllody phytoplasmas and that it belongs to the Ca. P. aurantifolia (16SrII) group. This result was well supported by the phylogenetic analysis showing GePP1 (16Sr RNA, SecY, rp and SecA genes) closely clustering with the Ca. P. aurantifolia 16SrII group isolates reported so far. The virtual RFLP pattern generated for the phytoplasma from gerbera was different (similarity coefficient 0.89) from the reference pattern of Ca. P. aurantifolia (16Sr II) subgroup after analysis with four enzymes (BfaI, Hha1, Sau3AI and RsaI). Based on the threshold similarity coefficient for a new subgroup (delineation should be set at 0.97), the GePP1 may be considered as new subgroup of Ca. P. aurantifolia (16SrII) group. This is the first report of Ca. P. aurantifolia belonging to 16Sr II group affecting gerbera in India. Keywords: Candidatus Phytoplasma aurantifolia; phyllody; gerbera; PCR; phylogenetic analysis.

Occurrence, Distribution and Molecular Characterization of Phytoplasma Infecting Chickpea (Cicer arietinum L.) from Tamil Nadu

Phytoplasma is an obligate prokaryote infecting a wide array of crops such as urdbean, sesame, brinjal and many other vegetable crops in India. Several workers have noticed the recent outbreak of phytoplasma disease in several pulse crops. A study was conducted to identify the phytoplasma infection in chickpea from Tamil Nadu. The average disease incidence due to phytoplasma was recorded with a range of 5-20% under field condition. The infected plants produced symptoms including stunting of plants, bushy appearance, reduced leaf size, chlorosis and reddening of infected leaves. The association of phytoplasma with these symptoms was confirmed by nested PCR assay using the universal primers P1/P7 and R16F2n/R16R2. The infected samples were amplified with an amplicon size of 1.2 kb and sequence analysis showed more than 99% similarity with phytoplasma belongs to Candidatus Phytoplasma aurantifolia. Phylogenetic analysis of nucleotide sequences confirmed the chickpea phyllody phytoplasma forms a single subgroup with other Indian isolates of various crops.

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.

Molecular detection and characterization of a 16SrII-D phytoplasma associated with streak yellows of date palm in Oman

Date palm is the most important fruit crop in Oman, covering about 50% of the agricultural area in the country. During a survey in 2018–2020, symptoms resembling phytoplasma diseases were observed in date palms in Muscat, Oman. Date palms showed streak yellows and dryness of young and mature leaflets and leaves. Polymerase chain reaction (PCR) assays were conducted using the universal primer pairs P1/P7 followed by R16F2n/R16r2n. In addition, the phytoplasma genes imp, secA, and tuf were also targeted in semi-nested PCR assays by using specific primers for each gene. Symptomatic trees were positive for phytoplasma presence in nested and semi-direct PCR, however, no amplification was observed from the symptomless and negative control. Sequence analysis of the rRNA, imp, secA, and tuf genes confirmed that the phytoplasma associated with streak yellow date palm (SYDP) is a strain of the 16SrII-D subgroup. This is the first record of association of a phytoplasmas of the 16SrII-D subgroup with date palms in Oman. This finding increased our knowledge about the distribution of phytoplasma diseases in date palms in the world.

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.

Association of the 16SrII-D Phytoplasma with African Marigold (Tagetes erecta) Phyllody in Oman.

The African marigold (Tagetes erecta L.) is an ornamental, herbaceous plant commonly found in Oman. In 2019, African marigold plants showing phyllody and virescence symptoms, which are typical symptoms of phytoplasmas disease, were found in at Sultan Qaboos University in Oman. Transmission electron microscopy of marigold leaf midrib from phyllody disease plants showed the presence of numerous phytoplasma bodies in the sieve tube of all of the symptomatic samples. DNA was extracted from asymptomatic and symptomatic marigold plant samples, followed by PCR of the 16S ribosomal RNA (rRNA) and imp genes. The PCR assays showed that the symptomatic plants are positive for phytoplasma. The DNA sequence analysis and phylogenetic trees showed that the 16S rDNA and imp gene sequences from all marigold phyllody strains shared 100% sequence identity to 16SrII-D subgroup sequences in the GenBank. This is the first report of a phytoplasma of the 16SrII-D subgroup associated with the African marigold (T. erecta) worldwide.

First Report of Association of 16SrII-D Phytoplasma with Cycas revoluta in Oman

First Report of Association of 16SrII-D Phytoplasma with <i>Cycas revoluta</i> in Oman

Evidence for the association of mastrevirus, cucumovirus and phytoplasma with chickpea stunt disease and their putative insect vectors in India

Symptoms of severe stunting, little leaf, yellowing and axillary shoot proliferation were recorded on chickpea plants in three northern states of India (New Delhi, Uttar Pradesh and Rajasthan) during January–March, 2017–2018 with disease incidences of 6–56%. Nested PCR assays with phytoplasma-specific universal primer pairs (P1/P7 and 3Far/3Rev) and 16S rRNA gene sequence analysis confirmed the association of ‘Candidatus Phytoplasma trifolii’ in the symptomatic chickpea variety Pusa Bheema at New Delhi. The in silico RFLP and phylogeny analyses of 16S rRNA sequence of the chickpea stunt (CpS) affected samples confirmed the association of 16SrVI-D subgroup phytoplasma. Similar phytoplasma strain (16SrVI-D subgroup) was detected in the leafhopper species, Hishimonus phycitis collected from symptomatic chickpea fields at New Delhi. Association of icosahedral virus particles of ~25–30 nm diameters were seen under electron microscope in CpS affected samples as well as in the aphids, Myzus persicae (Sulzer) collected from the symptomatic CpS samples at New Delhi. Both the plant and aphid samples were tested positive with the polyclonal antibodies (pAbs) of cucumber mosaic virus (CMV) in DAS-ELISA and ISEM tests. PCR assays using coat protein (CP) gene specific primers for CMV and the resultant sequence comparison also confirmed the association of CMV with the CpS affected symptomatic chickpea plants and M. persicae. The CpS affected samples collected from all the three states were further tested positive with chickpea chlorotic dwarf virus (chickpea chlorotic dwarf virus, CpCDV) CP gene specific primers in PCR assays. The 2.7 kb DNA sequence resulted from the rolling circle amplification (RCA) from the CpS affected chickpea samples of New Delhi revealed a highest sequence similarity of 99% with the CpCDV isolate from Pakistan. These results suggested a wider occurrence of CpCDV in chickpea crops in all the three states and a mixed infection of phytoplasma [‘Ca. P. trifolii’ (16SrVI-D subgroup)] and two plant viruses (CMV and CpCDV) associated with CpS disease at New Delhi.

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.

First Report of a ‘Candidatus Phytoplasma aurantifolia’ Strain Associated with Virescence, Floral Proliferation, and Dwarf Symptoms on Indigofera suffruticosa in Taiwan

Indigofera suffruticosa Mill., generally known as anil or Guatemalan indigo, is a perennial shrub planting in tropical and subtropical regions, including Taiwan; it was commonly used to produce the indigo dye, which is still being used in many personal workshops in Taiwan. There were a few reported cases of the association of phytoplasma with Indigofera spp. In 1983, I. suffruticosa showing phyllody and small leaf symptoms was first described, but no molecular identification was conducted (Albanese et al. 1995). Indigofera linifolia with little leaf, phyllody, and proliferation symptoms and Indigofera colutea showing little leaf symptoms in Australia caused by phytoplasma strains were reportedly associated with papaya yellow crinkle disease (Padovan and Gibb 2001). Affected Indigofera juncea and Indigofera hirsuta exhibiting witches’-broom symptoms were reported to be the same as the sesame phyllody phytoplasma isolate in Thailand (Nakashima et al. 1995). In February 2016, I. suffruticosa plants showing virescence, floral proliferation, and dwarf symptoms were collected from commercial fields of Mingjian Township, Nantou County, Taiwan. Two each of symptomatic and asymptomatic samples were collected from the field for further study. Transmission electron microscopic observation revealed the presence of the pleomorphic phytoplasma cells only in the phloem sieve cells of the two symptomatic I. suffruticosa plants. The two asymptomatic and two symptomatic samples were used for total DNA extraction with the modified CTAB method, and the amplicons of the nested PCR with universal primer pairs for nearly full-length 16S rDNA amplification (P1/P7 followed by R16F2n/R16R2) were electrophoresed in 0.8% agarose gel (Fulton et al. 1995; Lee et al. 1998). A 1.2-kb fragment (GenBank accession no. KX268346) was identified only from the two symptomatic I. suffruticosa plants. Analysis of 16S rDNA sequences by iPhyClassifier indicated that the phytoplasma associated with virescence symptoms in I. suffruticosa shared a 98.2% identity with that of the ‘Candidatus Phytoplasma aurantifolia’ reference strain (GenBank accession no. U15442). Virtual restriction fragment length polymorphism patterns were identical to those of the 16SrII-V subgroup (Zhao et al. 2009). Taken together, the results indicated that the phytoplasma associated with I. suffruticosa in Taiwan is a ‘Ca. P. aurantifolia’-related strain. The occurrence of phytoplasma in I. suffruticosa could have direct implication on economic profit for growers and the handicraft industry in Taiwan. To our knowledge, this is the first report of a 16SrII-V subgroup phytoplasma causing virescence, floral proliferation, and dwarf symptoms on I. suffruticosa in Taiwan.