Phyllody Phytoplasma Research Articles

Tephrosia purpurea Represents a New Host of 16SrII-V Subgroup Phytoplasma Associated with Witches'-Broom Disease in China.

Tephrosia purpurea is a medical plant with excellent insecticidal activity belonging to the family of Leguminosae distributed throughout southern of China (Pei et al., 2013). During January to February 2021, the plants showing abnormal symptoms including witches'-broom, internode shortening, leaf chlorosis and leaflet formation, as shown in Fig.1, were found in Ledong County of Hainan Province, a tropical island in China, with about 60 % incidence. The Tephrosia purpurea disease symptoms were suspected to be induced by phytoplasma, a phloem-limited prokaryotic pathogen which can not be cultured in vitro and which causes severe financial loss and ecological damage to the island. Total DNA from the symptomatic and asymptomatic samples of Tephrosia purpurea were extracted using 0.10 g fresh plant leaves and branches by CTAB method (Doyle and Doyle, 1990). 16S rRNA and secA gene sequence fragments of phytoplasma were detected through PCR amplification using primers R16mF2/R16mR1 (Gundersen and Lee, 1996) and secAfor1/secArev3 (Hodgetts et al., 2008). The two gene sequence fragments of phytoplasma were obtained from the DNA of six symptomatic plant samples whereas not from the DNA of six asymptomatic plant samples. These amplified products were sequenced and the data were deposited in GenBank. The two gene sequence fragments of the DNA obtained from the diseased plant samples were all identical, with a length of 1335 bp for the 16S rRNA (GenBank accession: MW616560) and 729 bp for the secA gene (MW603929). The secA gene fragment putatively encodes for 242 amino acids. The phytoplasma strain was named as Tephrosia purpurea witches'-broom (TpWB) phytoplasma, TpWB-hnld strain. 16S rRNA gene sequence fragment of TpWB-hnld was analyzed by online tool iPhyClassifier (Wei et al., 2007), indicating that the pathogen strain was a member of subgroup 16SrII-V and a 'Candidatus Phytoplasma aurantifolia'-related strain. Blast analysis based on the 16S rRNA gene sequence fragment of TpWB-hnld showed 100 % sequence identity with that of peanut witches'-broom group members (16SrII group), such as Cassava witches'-broom phytoplasma (KM280679) and Cleome sp. phytoplasma (KM280677); Blast analysis based on the secA gene sequence fragment of TpWB-hnld showed 100 % sequence identity with that of peanut witches'-broom group members (16SrII group), such as sesame phyllody phytoplasma (JN977044). Homology and phylogeny were analyzed using the software of DNAMAN 5.0 and MEGA 7.0, indicating that TpWB-hnld and other subgroup 16SrII-V phytoplasma strains, including Cassava witches'-broom phytoplasma, Cleome sp. phytoplasma, Crotalaria witches'-broom phytoplasma (EU650181) and Desmodium ovalifolium witches'-broom phytoplasma (GU113152), were clustered into one clade with 98 % bootstrap value based on the 16S rRNA gene sequence fragments; TpWB-hnld and sesame phyllody phytoplasma were clustered into one clade based on the secA gene sequence fragments. Multiple alignment based on the 16S rRNA gene sequence fragment showed that the TpWB-hnld phytoplasma strain showed 98 % sequence identity with TpWB phytoplasma strain (HG792252) belonging to 16SrII-M subgroup reported in India (Yadav et al., 2014). To our knowledge, this was the first time that 16SrII-V subgroup phytoplasma associated with Tephrosia purpurea witches'-broom disease was identified in China. Molecular analysis based on the 16S rRNA and secA gene sequence fragments indicated that TpWB-hnld phytoplasma was a member of subgroup 16SrII-V and a 'Candidatus Phytoplasma aurantifolia'-related strain.

Loop-mediated isothermal amplification (LAMP) assay for detection of sesame phyllody phytoplasmas in Vietnam.

Phloem-limiting phytoplasmas are known to be causal agents of phyllody, which is recognized by the abnormal development of floral structures resulting in serious yield losses in sesame plants. Currently, identification of the various groups of phytoplasmas that cause sesame phyllody (SP) is conducted by nested PCR, RFLP, and multiplex real-time qPCR assays. However, these methods require intensive labor and are costly and time-consuming so can only be undertaken in well-equipped labs. Here, diagnostic loop-mediated isothermal amplification (LAMP)-based assays allowing rapid detection of specific groups of phytoplasmas within 30min were developed based on detection of the 16S rRNA sequence of phytoplasmas. Universal 16S rRNA phytoplasma primers and seven primer sets of different 16Sr group phytoplasmas (16SrI, 16SrII, 16SrIII, 16SrIV, 16SrV, 16SrX, 16SrXI) and universal plant cytochrome oxidase (cox) gene primers were used to detect 16S rRNA group phytoplasma sequences and the cox gene in sesame plants. The LAMP assays were carried out using a real-time fluorometer with amplification plots and annealing curves visualized directly. Results demonstrated that the 16SrI and 16SrII group phytoplasmas were causal agents of sesame phyllody in Vietnam. LAMP-based assays for in-field detection of sesame phyllody-causing phytoplasmas revealed advantages and potential applicability in comparison with conventional approaches. To the best of our knowledge, this is the first assessment of multiple phytoplasma infection associated with sesame phyllody disease in Vietnam using LAMP-based assays.

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.

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.

First report of the association of a ‘Candidatus Phytoplasma asteris’ strain with Crossandra infundibuliformis

Crossandra infundibuliformis (firecracker flower, Acanthaceae) is an evergreen, perennial, flowering plant which is native to South India and Sri Lanka, and valued medicinally (Vadivel & Panwal, 6). Phytoplasmas are cell-wall-less unculturable prokaryotes transmitted by phloem-sucking leafhoppers and planthoppers that cause diseases in over a thousand plant species, including crop, ornamental and native plants worldwide (Rao et al., 4). So far, there are no records of phytoplasmas infecting C. infundibuliformis. Crossandra infundibuliformis plants showing symptoms of leaf yellowing were observed in the Devanahalli area of Bengaluru, India during June 2016 (Figs. 1-2); 30-40% of the plants surveyed in an area of c. 0.2 hectares had symptoms. Leaf samples were collected from three diseased and three symptomless C. infundibuliformis plants and DNA was extracted from leaf midribs using the DNeasy plant mini kit (Qiagen, Germany). The extracted DNA was used as a template in nested PCR with primer pairs, P1/P7 (Schneider et al., 5) and R16F2n/R16R2 (Gundersen & Lee, 1) that amplify the 16S rRNA gene, and primers SecAfor1/SecArev3 and SecAfor2/SecArev3 (Hodgetts et al., 2) which target the protein translocase subunit SecA (secA) gene. DNA extracted from the sesame phyllody phytoplasma (16SrI group; GenBank Accession No. KC920747) was used as a positive control. The ~1.2 kb and ~480 bp amplified products corresponding to the phytoplasma 16S rRNA and secA genes, respectively, were detected in all diseased C. infundibuliformis (CiLY) but not from the symptomless samples. Both 16S rDNA and secA amplicons were purified (WizardR SV Gel and PCR Cleanup System; Promega, USA), cloned (pGEM-T Easy Vector, Promega) and sequenced (ABA Biotech, India). The 16S rRNA (MT474158, MT474159) and secA (MT472835, MT472836) gene sequences from the symptomatic plants were deposited in GenBank. Sequence comparison and phylogenetic analysis (MEGA 7.0) of the 16S rRNA and secA gene sequences indicated that both shared 100% identity with members of the 16SrI phytoplasma group (formerly Aster yellows, ‘Candidatus Phytoplasma asteris‘), and clustered with 16SrI phytoplasma group-related strains (Figs. 3-4). The iPhyClassifier analysis based on the 16S rDNA F2nR2 sequence of the CiLY phytoplasma (https://plantpathology.ba.ars.usda.gov) yielded similar RFLP patterns to those of 16SrI reference strains, subgroup B (M30790), with a similarity coefficient of 1.0. The virtual RFLP results suggested that the CiLY phytoplasma belongs to the 16SrI-B subgroup. In India, ‘Ca. P. asteris’ is the most widespread phytoplasma group, identified from 64 plant species (Rao et al., 4). In previous studies, C. infundibuliformis plants exhibiting leaf yellowing symptoms were surveyed for phytoplasmas (Khasa et al., 3) but none were found. The present study is the first report of a phytoplasma associated with a leaf yellowing disease in C. infundibuliformis. The results have a significant phytosanitary impact for the epidemiology of the 16SrI phytoplasma group for the Acanthaceae family and related species, as well as, for 16SrI phytoplasma associated diseases in India and the region. The authors wish to express sincere thanks to the Head of Division of Plant Pathology and Director of the Indian Agricultural Research Institute for providing laboratory facilities.

First report of ‘Candidatus Phytoplasma aurantifolia’(16SrII-A) associated with strawflower phyllody

Four strawflower plant samples showing phyllody disease symptoms were collected from the experimental plot at the College of Horticulture, Bangalore, Karnataka (India). The phytoplasma infection in strawflower was confirmed through PCR using 16Sr RNA and SecY gene specific primers. All four strawflower plant samples gave positive amplification 16Sr RNA (1.8 kb) and SecY gene (1.6 kb) for the phytoplasma. The amplified products were cloned and sequenced. The sequences of four clones obtained were identical. Therefore, a representative isolate (SWF1) nucleotide (nt) sequence of 16Sr RNA and SecY gene comparisons were made with the available phytoplasmas 16Sr RNA and SecY gene nt sequences from NCBI database. The analysis showed that strawflower phyllody phytoplasma shared the highest identity of 97.3 to 98.5% (16S rRNA) and 99.4 to 99.5% (SecY gene) with ‘Candidatus Phytoplasma aurantifolia’ group (16SrII) isolates reported from different parts of the world. This was well supported by close clustering of strawflower phytoplasma in the current study with ‘Ca. P. aurantifolia’ 16SrII groups in the phylogenetic analysis. The virtual RFLP pattern generated for the phytoplasma from strawflower phyllody was similar (similarity coefficient 0.97) to the reference pattern of 16Sr group II and subgroup A (‘Ca.P. aurantifolia’, 16SrII). This is the first report of phytoplasma associated with the phyllody disease of strawflower in India.

First report of the association of a ‘Candidatus Phytoplasma asteris‘‐ related strain with Plumbago auriculata leaf yellowing in India

Plumbago auriculata (Plumbaginaceae) is a perennial ornamental shrub with many therapeutic values and native to South Africa (Saji & Antony, 5). Several groups of phytoplasma have been reported to infect ornamental and medicinal plants all over the world (Rao et al., 2). However, no phytoplasma has been found so far in P. auriculata. Leaf yellowing symptoms on P. auriculata plants (Fig. 1) were observed in Baramati, Pune, India in March 2016 with a disease incidence of 8-10%. To verify the presence of phytoplasma, three symptom-bearing and three asymptomatic P. auriculata samples were collected and DNA was extracted using the CTAB protocol. PCR was performed in a Mastercycler (Eppendorf, Germany) (Rao et al., 4) and DNA from the sesame phyllody phytoplasma (16SrI group) (GenBank Accession No. KC920747) was used as a positive control. The 16S rRNA gene was amplified from all the symptomatic P. auriculata leaf yellowing (PaLY) samples but not from the asymptomatic samples using P1/P7 (Schneider et al., 6) followed by R16F2n/R2 primer pairs (Gundersen & Lee, 1) in nested PCR. Amplicons of the expected size (c. 1.25 kb) were purified and directly sequenced (MN239503 and MN239504). BLAST analysis showed that these 16Sr RNA gene sequences shared 100% identity with phytoplasmas in the16SrI group (MG252367, MK440284, MK440282, KX15181 & KT957205). A phylogenetic tree was constructed using the neighbour-joining method with MEGA 7.0 (Fig.2). The R16F2n/R2 sequence of the PaLY phytoplasma was subjected to in silico RFLP using the iPhyClassifier online tool (https://plantpathology.ba.ars.usda.gov). The iPhyClassifier analysis indicated that the virtual RFLP patterns derived from the 16S rDNA F2n/R2 fragment of the PaLY phytoplasma strain with restriction enzymes were similar to those of a phytoplasma strain from group 16SrI, ‘Candidatus Phytoplasma asteris’ (M30790), formerly Aster Yellows Group, subgroup B, with a similarity coefficient of 1.0. Based on sequence and RFLP results, the PaLY phytoplasma is classified as a member of the phytoplasma subgroup 16SrI-B. Phytoplasmas have been recorded from plant species in the Plumbaginaceae such as Limonium sinuatum in Canada, Europe and Israel (Rao et al., 2). In India, the ‘Ca. P. asteris’ group is the most widespread group and has been found in 64 plant species (Rao et al., 3). Phytoplasmas in subgroup 16SrI-B have been associated particularly with diseases in pineapple, sesame, sugarcane, squash, rose and fennel. The present study reports P. auriculata as a new host for the 16Sr I-B phytoplasma subgroup worldwide. The authors wish to express sincere thanks to Head, Division of Plant Pathology and Director, Indian Agricultural Research Institute for providing laboratory facilities.

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.

First Report of Phytoplasma Infecting Pterocarpus indicus in China

Pterocarpus indicus Willd., an adaptable and fast-growing landscape tree, is commonly cultivated in landscaping in southern China. In April 2018, a P. indicus tree with symptoms of flat branches and typical fasciation, which had the same symptoms of phytoplasma infection, was observed in Haikou, Hainan province, China. To determine the presence of phytoplasma, stem samples of symptomatic and asymptomatic P. indicus were subjected to total DNA extraction using the plant Genomic DNA Kit (Tiangen, Beijing, China), followed by nested polymerase chain reaction (PCR) detection of phytoplasma (Jomantiene et al. 1998; Lee et al. 1998). Briefly, the universal primer pair R16mF2/R16mR1 was employed for the initial PCR amplification of 16SrDNA fragment of phytoplasma and primer set R16F2n/R16R2 (Lee et al. 1998) for nested PCR amplification. As expected, the PCR products were generated from the samples of the symptomatic plant but not from the symptomless control samples. The anticipated amplicon was approximately 1.2 kb in size. The PCR products were gel purified using TIANgel Midi Purification Kit (Tiangen, Beijing, China) and cloned into pMD-18T vector (Takara Bio, Dalian, China). Three independent positive clones were subjected to Sanger sequencing. The obtained sequence was deposited into NCBI GenBank database with GenBank accession no. MH727702. A BLASTn search against the nonredundant nucleotide database revealed high sequence identities with the corresponding regions of phytoplasmas of aster yellows phytoplasma group (16SrI). A maximum of 99.7% nucleotide sequence identity with Malvastrum coromandelianum phyllody phytoplasma (GenBank accession no. MF490802) was observed. Moreover, an iPhyClassifier (Zhao et al. 2013) operation was used to perform sequence comparison and generate a virtual restriction fragment length polymorphism (RFLP) profile for the sequence derived from the symptomatic P. indicus sample, which showed the most similarity with that of the reference strain (GenBank accession no. AP006628) belonging to 16SrI group. However, this strain may represent a new subgroup because the similarity coefficient was only 0.91, which is within the values set for a new subgroup (Biswas et al. 2013). To our best knowledge, this is the first report of phytoplasma infecting P. indicus in China.

Non-crop sources of Rapeseed Phyllody phytoplasma (‘Candidatus Phytoplasma asteris’: 16SrI-B and 16SrI-(B/L)L), and closely related strains

In Poland, a phytoplasma belonging to the subgroup 16SrI-B of ‘Ca. Phytoplasma asteris’ species causes disease in winter oilseed rape (Brassica napus), named rapeseed phyllody (RP). Affected plants exhibit malformations of flowers – phyllody, leading to losses in seed production. However, the potential sources of RP phytoplasma (RPp) outside oilseed rape are poorly understood. A screening survey of non-crop wild plants associated with winter oilseed rape was performed in order to identify alternative plant hosts of RPp. Nested polymerase chain reaction (PCR) and sequencing of PCR-amplified 16S rDNA fragments were employed for the detection and identification of phytoplasma strains affecting oilseed rape crops and adjacent wild plants. Phytoplasmas belonging to subgroups 16SrI-B, 16SrI-L, 16SrI-(B/L)L and 16SrI-C, as well as subgroup 16SrXII-H, were detected in non-crop plants. All investigated oilseed rapes manifesting RP disease were positive to ‘Ca. Phytoplasma asteris’ ribosomal subgroup 16SrI-B or heterogenic variant 16SrI-(B/L)L. Wild plants species were predominantly infected with the same phytoplasma subgroups as oilseed rapes. However, comparative sequence analysis revealed single nucleotide polymorphisms that indicate divergence of 16SrI-B and 16SrI-(B/L)L phytoplasma strains in non-crop species. Surprisingly, most of the weeds which tested positive for phytoplasma did not exhibit any characteristic symptoms of infection. In this study we identified for the first time perennial (Elymus repens, Lolium perenne) and annual (Echinochloa crus-galli, Polygonum aviculare, Viola arvensis, Matricaria perforata, Stelaria media) plants as asymptomatic reservoirs of RPp related strains in the winter oilseed rape agroecosystem. We also confirmed RPp related infections in diseased Apera spica-venti, Sisymbrium officinale, Matricaria discoidea, and Papaver rhoeas. These plants potentially play a significant role in RPp evolution and disease aetiology and epidemiology.

Identification of sesame phyllody transmitting insect vectors in Assam, India

Different leafhopper species transmitting sesame phyllody phytoplasma were reported. Leafhoppers were collected from sesame phyllody infected field at different time interval and their population was recorded. Four leafhopper species were predominantly observed in the field conditions: Hishimonus phycitis, Exitianus indicus, Cofana unimaculata and Nephotettix nigropictus. Transmission studies were carried out by giving an acquisition and inoculation feeding period of 7 days each. Among all the leafhoppers, H. phycitis was found to transmit sesame phyllody phytoplasma (83.33%) to healthy sesame plants as confirmed by PCR and nested PCR assays followed by sequencing. The BLAST analysis of 16S rDNA sequences from H. phycitis confirmed the phytoplasma presence indicating that is clusters with the aster yellows phytoplasmas.

Detection of a possible insect vector and alternative host plants for the sesame phyllody phytoplasma in Assam, India

Detection of a possible insect vector and alternative host plants for the sesame phyllody phytoplasma in Assam, India

English

English

First Report of ‘Candidatus Phytoplasma asteris’ Related Strain Associated with Atractylodes lancea Malformations in Hubei Province of China

HomePlant DiseaseVol. 102, No. 12First Report of ‘Candidatus Phytoplasma asteris’ Related Strain Associated with Atractylodes lancea Malformations in Hubei Province of China PreviousNext DISEASE NOTES OPENOpen Access licenseFirst Report of ‘Candidatus Phytoplasma asteris’ Related Strain Associated with Atractylodes lancea Malformations in Hubei Province of ChinaQ. Wan, L. Chen, S. Ming, L. Gong, X. Huang, K. Yu, H.-Y. Che, and D.-Q. LuoQ. WanSearch for more papers by this author, L. ChenSearch for more papers by this author, S. MingSearch for more papers by this author, L. GongSearch for more papers by this author, X. HuangSearch for more papers by this author, K. Yu†Corresponding author: K. Yu; E-mail: E-mail Address: [email protected]http://orcid.org/0000-0002-4262-4431Search for more papers by this author, H.-Y. Chehttp://orcid.org/0000-0002-9302-7784Search for more papers by this author, and D.-Q. LuoSearch for more papers by this authorAffiliationsAuthors and Affiliations Q. Wan L. Chen S. Ming L. Gong X. Huang K. Yu † , Hubei Key Laboratory of Resources and Chemistry of Chinese Medicine, College of Pharmacy, Hubei University of Chinese Medicine, Wuhan, China H.-Y. Che D.-Q. Luo , Environment and Plant Protection Research Institute, Chinese Academy of Tropical Agriculture Science, Hainan, China. Published Online:10 Oct 2018https://doi.org/10.1094/PDIS-01-18-0177-PDNAboutSections ToolsAdd to favoritesDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InRedditEmailWechat Atractylodes lancea Thunb., a commercially cultivated plant in Hubei, Shanxi, and Henan Provinces, China, is well known for its extensive use in the practice of traditional Chinese medicine for the treatment of various diseases (Huang et al. 2016). During the investigations conducted in April 2017 in Yingshan (30°30′ to 31°08′ N; 115°32′ to 116°04′ E), Hubei Province, 100 plants of A. lancea with symptoms including stem fasciation and proliferation were noticed across the five surveyed fields. Other symptoms including floral virescence and inflorescence abnormality were also observed. Stem tissues were sampled from six symptomatic and three asymptomatic A. lancea plants, and molecular diagnostics were employed to prove possible phytoplasma infections. Total DNA was extracted using the Plant Genomic DNA Kit (Tiangen, Beijing, China) following the manufacturer’s instructions, and nested polymerase chain reaction (PCR) was performed with P1/P7 followed by R16F2n/R16R2 primer pairs (Lee et al. 1998). A phytoplasma-specific gene fragment of approximately 1,200 base pairs (bp) was amplified from all symptomatic plants. In parallel diagnostic assays, no amplicons were generated from asymptomatic plants. The amplicon of approximately 1,200 bp of the second PCR product from symptomatic samples was purified, cloned into pMD-18T vector (Takara Bio, Dalian, China), and sequenced. The sequences obtained from all six symptomatic plants were identical, and one of them was deposited in the GenBank under accession number MG257945.3. An NCBI-BLAST search indicated that it shared 99.7% identity to the sesame phyllody phytoplasma (GenBank accession no. KF728953) from India. A phylogenetic tree of phytoplasma 16S rRNA gene sequences was constructed using a neighbor-joining algorithm, and it showed that A. lancea isolates clustered with the phytoplasma strains belonging to subgroup 16SrI-B (GenBank accession nos. MG652627 and KY565572). Restriction fragment length polymorphism analyses using iPhyClassifier (Zhao et al. 2009) placed the A. lancea phytoplasma strain in the 16SrI-B subgroup because the virtual pattern was identical (similarity coefficient 0.92) to the reference pattern of 16Sr group I, subgroup B (CP000061). Although ‘Ca. Phytoplasma asteris’ 16SrI-B subgroup has been identified in various hosts (Alvarez et al. 2014), to our knowledge, this is the first report of 16SrI-B subgroup phytoplasma infecting A. lancea in China. With its rapid and widespread characteristics, the identified phytoplasma strain could pose an imminent threat and cause economic loss to A. lancea cultivation. A. lancea is widely distributed and cultivated also in Japan and Thailand (Na-Bangchang and Karbwang 2014), and a phytoplasma, detected in China, could be a potential problem for all the region and seriously affect the production of rhizomes of A. lancea, which have a great pharmacological value worldwide.

Detection and characterisation of a phytoplasma associated with cucumber (Cucumis sativus) regional yellows disease in Iran

A phytoplasma was detected in cucumber (Cucumis sativus), exhibiting regional yellows symptoms in leaves, stem and fruits, that was grown in the greenhouse near Tehran (Iran). Since this is a previously undescribed disease, the name cucumber regional yellows have been tentatively assigned to it. Based on in silico RFLP and phylogenetic analysis of PCR-amplified 16S rDNA sequences, the phytoplasma associated with regional yellows disease was identified as a new member of phytoplasma 16S rRNA group VI (16SrVI-A) with closest relationships to zucchini phyllody phytoplasma (KP119494). According to our results, cucumber regional yellows phytoplasma could be designated as a subgroup VI-A.

First report of a ‘Candidatus Phytoplasma trifolii‘‐related strain associated with rapeseed witches' broom in Iran

Rapeseed (Brassica napus) is an economically important oilseed crop grown in many countries worldwide. During recent years rapeseed cultivation has developed in regions of Iran. In April 2018, symptoms similar to phytoplasma-associated diseases including witches' broom (Fig. 1), stunting and little leaves (Fig. 2) were observed in rapeseed in several areas of Hormozgan province, Iran. To identify the association of phytoplasma with the diseased plants, total genomic DNA was extracted from midribs of eight symptom-bearing and two symptomless plants using the CTAB method. The samples were analysed for phytoplasma DNA by direct PCR using the phytoplasma universal primer pair P1/P7 and nested PCR using primers P1/P7 (first round) followed by R16F2n/R16R2 (second round) (Gundersen & Lee, 2). Amplicons of the expected sizes (1800 and 1250 bp) were amplified with direct and nested PCR in all symptom-bearing plants but not from symptomless plants or sterile distilled water used as negative controls. A positive nested PCR product was directly sequenced and deposited in GenBank under accession no. MH430092. A BLAST search using the sequenced fragment showed that the phytoplasma associated with rapeseed witches' broom (RsWB) had 100% nucleotide identity with the corresponding sequence of several members (KX773529, MG788318 and AM260486) of the 16SrVI phytoplasma group and 99.8% identity with the type strain, ‘Candidatus Phytoplasma trifolii’ (AY390261). The virtual RFLP revealed that the RsWB phytoplasma belonged to the 16SrVI-A subgroup. There are several reports regarding the association of aster yellows group (16SrI) phytoplasmas with rapeseed (e.g. rapeseed phyllody phytoplasma) in Iran (Salehi et al., 6) and other countries (Maliogka et al., 4; Olivier et al., 5). To our knowledge, this is the first report of natural infection of rapeseed by a 16SrVI phytoplasma in Iran and most probably in the world. Phytoplasmas belonging to the 16SrVI-A have been previously reported on several economically vegetable crops such as tomato and cucumber in Iran (Esmailzadeh Hosseini et al., 1; Jamshidi et al., 3). Therefore, the infected volunteer rapeseed around tomato and cucumber fields may act as an alternative host for this phytoplasma. The authors would like to thank Dr. Majid Siampour for his excellent technical assistance.

Detection and identification of a ‘Candidatus phytoplasma aurantifolia’-related strain (16SrII-A subgroup) associated with corchorus aestuans phyllody in China

In September 2015, a phyllody that is typical of phytoplasma infection was observed on Corchorus aestuans plants in Haikou, Hainan Province, China. Total DNA from symptomatic and asymptomatic plants was extracted for molecular diagnosis. On the basis of sequence analysis and phylogenetic trees based on 16S rDNA and rp genes, the phyllody phytoplasma was ascertained to be related to ‘Candidatus phytoplasma aurantifolia’. To the best of our knowledge, this is the first report of a phytoplasma infecting C. aestuans in the world.

Identification of phytoplasmas from Neoaliturus haematoceps associated with sesame phyllody disease in southwestern Turkey

AbstractPhytoplasmas were detected based on nested PCR of the F2nR2 region of the 16S rDNA from Neoaliturus haematoceps (Mulsant and Rey) (Family: Cicadellidae). A total of 65 insect samples collected from sesame fields in Antalya, Turkey, during 2012–2014 were tested for phytoplasma detection. Phytoplasmas detected in fifteen samples showed an amplicon approximately 1250 bp in size using the universal primers of P1/P7 and R16F2n/R16R2. Identification of the phytoplasmas by sequence analysis revealed three different 16S rDNA phytoplasma groups: the peanut witches’‐broom, group II; clover proliferation, group VI; and pigeon pea witches’‐broom, group IX. The molecular characterization of subgroups was determined by sequence analysis and PCR‐RFLP using the restriction enzymes RsaI and TaqI. Restriction profiles of the subgroups were also confirmed using the iPhyclassifier program. BLAST and PCR‐RFLP analyses classified the subgroups as II‐D, VI‐A and IX‐C. This is the first report of molecular detection of three 16S rDNA subgroups of phytoplasmas, II‐D, VI‐A and IX‐C, from N. haematoceps in Turkey. This study also supports earlier studies of sesame phyllody phytoplasmas by N. haematoceps.