Group 16SrI Research Articles

First report of association of 'Candidatus Phytoplasma asteris' with Moringa oleifera leaf yellowing and stunting disease in India.

Moringa oleifera (family Moringaceae) also known as the 'drumstick tree' is a significant nutritious and medicinal plant that is commonly grown in India and contains a variety of vital phytochemicals. M. oleifera is used in several Indian herbal medicine formulations to treat a variety of illnesses (Kumar and Rao 2021). Typical phytoplasma symptoms of leaf yellowing and stunting were observed in M. oleifera trees up to 10% incidence at Acharya Narendra Dev University of Agriculture & Technology, Ayodhya, Uttar Pradesh, India in November 2021 and stunting with less fruit bearings symptoms with 8% incidence in October 2021 at Jonnalakothapalle village of Mudigubba mandal of Ananthapuramu district in Andhra Pradesh, India (Fig.1a, b). To investigate the possibility of a phytoplasma association with the symptoms, total DNA was isolated from the leaf samples collected from two diseased and two healthy plants from both the locations using CTAB method. The DNAs isolated were analysed by nested polymerase chain reaction (PCR) with universal phytoplasma primer pairs P1/P7 and R16F2n/R16R2 for the 16S rRNA gene (Deng and Hiruki 1991; Gundersen and Lee 1996) and secAfor1/sArev3 and SecAfor2/ SecArev3 for secA gene (Hodgetts et al. 2008). Amplicons of the expected size (~1.25kb from 16S rRNA gene and ~480bp from secA gene) were obtained from symptomatic plants only. The nested PCR products were cloned (pGEM-T Easy Vector, Promega), sequenced (ABA Biotech, India) and the sequences were deposited in GenBank with accession numbers OP358449, OP358450, OP358451, OP358452 for the 16SrRNA gene (~1.25 kb) and OP358443, OP358444, OP358445, OP358446 for the secA gene (~480 bp). BLASTn analysis revealed that the partial 16S rRNA gene sequences of M. oleifera phytoplasma isolate shared up to 99.9% sequence identity with the strain 'Candidatus Phytoplasma asteris' (Accession numbers MN909051, MN909047) and secA gene sequences shared up to 100% sequence identity with 'Ca. Phytoplasma asteris' (Accession numbers KJ434315, KJ462009) belonging to 16SrI group. The 16S rRNA and secA genes sequence-based phylogenetic analysis (Figure 1d,e) showed that the phytoplasma strain associated with M. oleifera leaf yellowing and stunting clustered within the 16SrI phytoplasma group closest to 16SrI-B ('Ca. P. asteris') subgroup strains. Furthermore, the virtual RFLP pattern derived from the query 16S rDNA F2nR2 fragment is identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession: AP006628). To the best of our knowledge, this is the first report of the 16SrI-B subgroup of the phytoplasma strains with M. oleifera in the world. 'Candidatus Phytoplasma asteris' (16SrI-B subgroup) strains have been reported from several other commercial crops and weed hosts in India and efficient leafhopper vectors have been identified (Rao 2021; Reddy 2021). This indicates that the 'Ca. P. asteris'-related strains (16SrI-B) are widespread and infecting several plant species in India. The increasing incidence of the 16SrI-B strain and its wide host range in India strongly suggests further research into the epidemiology involved in the dynamic spread of the disease in order to recommend a suitable management approach.

Status of virus and phytoplasma diseases in carrot growing areas of Ankara and Konya provinces, Turkey

In this study, infections that may emerge from viruses and phytoplasmas were investigated in carrot cultivation fields in the Ankara and Konya provinces, which are located in the Central Anatolia Region of Turkey and have the largest agricultural areas of the country. A total of 272 symptomatic and asymptomatic carrot plants, together with their root and leaf parts, were collected from 61 fields by performing periodic field studies during the plant vegetation periods between 2018–2019. Samples were tested against carrot virus Y (CarVY), carrot red leaf virus (CtRLV), carrot red leaf virus-associated RNA (CtRLVaRNA), carrot mottle virus (CMoV), carrot torradovirus 1 (CaTV), carrot yellow leaf virus (CYLV), carrot thin leaf virus (CTLV) and cucumber mosaic virus (CMV) agents by Reverse Transcriptase-PCR (RT-PCR). To determine phytoplasmas, samples were tested using universal primers by nested PCR. Molecular tests conducted to investigate the aforementioned viruses produced negative results, but phytoplasma infections were widely found. 16SrVI-A “Candidatus Phytoplasma trifolii” and 16SrI-B “Aster yellows” subgroups were detected by in vitro/in silico RFLP and sequencing analyses. Disease incidence caused by both phytoplasma groups was 39.34%. This study determined for the first time that 16SrVI and 16SrI groups cause severe infections in the carrot fields of Turkey.

First report of 16SrII-V phytoplasma associated with cowpea (Vigna unguiculata) phyllody in China

Cowpea plants exhibiting virescence, phyllody and little leaf symptoms were observed in the fields of Zhanjiang City, Guangdong province of China in October 2020. Total DNA was extracted from symptomatic cowpea leaves. Using phytoplasma universal primer pairs (R16mF2/R16mR1, P1/P7) and a secA primer pair (secAfor1/secArev3), expected PCR fragments of approximately 1400, 1800 and 800 bp were amplified from all symptomatic cowpea leaves. MUSCLE analysis revealed that the representative 16S rRNA gene sequence of cowpea phyllody phytoplasma (CPP) shared 98.67% nucleotide identity with the reference strain WBDL of ‘Ca. Phytoplasma aurantifolia’ (GenBank accession number U15442); and 100% nucleotide identity with 16SrII-V phytoplasmas associated with ‘Praxelis clematidea’ phyllody (GenBank accession number KY568717); ‘Desmodium triflorum’ little leaf (GenBank accession number MT452308); and ‘Ixeris chinensis’, ‘Emilia sonchifolia’, and ‘Desmodium ovalifolium’ witches’ broom (GenBank accession number MT416114, MT420682 and MK956144, respectively). Phylogenetic analyses based on the 16S rRNA and secA gene sequences revealed that the CPP strain clustered with the 16SrII-V subgroup and 16SrII group, respectively. Virtual restriction fragment length polymorphism analysis of the 16S rRNA sequence revealed that CPP was associated with the 16SrII-V subgroup. To our knowledge, this is the first report of the16SrII-V subgroup of phytoplasmas associated with cowpea phyllody disease.

Molecular Detection of Phytoplasmas of the 16SrⅠ and 16SrXXXⅡ Groups in Elaeocarpus sylvestris Trees with Decline Disease in Jeju Island, South Korea

Phytoplasmas were discovered in diseased Elaeocarpus sylvestris trees growing on Jeju Island that showed symptoms of yellowing and darkening in the leaves. Leaf samples from 14 symptomatic plants in Jeju-si and Seogwipo-si were collected and phytoplasma 16S rRNA was successfully amplified by nested polymerase chain reaction using universal primers. The sequence analysis detected two phytoplasmas, which showed 99.5% identity to 'Candidatus Phytoplasma asteris' and 'Ca. P. malaysianum' affiliated to 16SrI and 16SrXXXII groups, respectively. Through polymerase chain reaction-restriction fragment length polymorphism (RFLP) analyses using the AfaI (RsaI) restriction enzyme, the presence of two phytoplasmas strains as well as cases of mixed infection of these strains was detected. In a virtual RFLP analysis with 17 restriction enzymes, the 16S rRNA sequence of the 'Ca. P. asteris' strain was found to match the pattern of the 16SrI-B subgroup. In addition, the phytoplasmas in the mixed-infection cases could be distinguished using specific primer sets. In conclusion, this study confirmed mixed infection of two phytoplasmas in one E. sylvestris plant, and also the presence of two phytoplasmas (of the 16SrⅠ and 16SrXXXⅡ groups) in Jeju Island (Republic of Korea).

Detection and molecular identification of a 16SrI group phytoplasma associated with sisal purple leafroll disease

Sisal purple leafroll disease (SPLD) is a destructive disease affecting sisal in China, however, its etiology remains unknown. This disease is characterized by purple margins and rolling and yellowing of the leaves. Recently, phytoplasma was found to be present in SPLD-affected sisal according to PCR assay. To further verify the presence of phytoplasma, determine its association with SPLD and identify the possible pathogenic phytoplasma infecting sisal, 80 symptomatic and 65 asymptomatic sisal samples were collected from the main sisal farms in three Chinese provinces, including Hainan, Guangdong and Guangxi provinces, from 2018 to 2021. PCR assays and transmission electron microscopy further revealed the presence of phytoplasma. Nested PCR using the universal primer R16mF2/R16mR1 followed by R16F2n/R16R2 was confirmed as the most effective molecular method for the detection of phytoplasma in sisal. A comparison of the detection rate of phytoplasma in symptomatic (83%) versus asymptomatic (17%) sisal populations showed some association between phytoplasma and SPLD. Sequencing analyses, phylogenetic analyses, and virtual restriction fragment length polymorphism analyses confirmed the presence of phytoplasma in sisal belonging to 16SrI-B. This is the first report of 16SrI-B phytoplasma infecting sisal in China.

First report of a ‘Candidatus Phytoplasma aurantifolia’ related strain (16SrII‐C) associated with Convolvulus virgatus witches'‐broom disease in Iran

Convolvulus virgatus (Convolvulaceae) is a perennial shrub which grows mainly in desert and shrubland biomes in Iran, the Arabian Peninsula and Pakistan (Wood et al., 2015). In a 2020 survey for phytoplasma diseases, witches'-broom disease of C. virgatus (WBDCV) was observed in the Roodan area (Hormozgan province, Iran). The main symptoms of WBDCV were flower and stem proliferation, shortened internodes, little leaf, severe witches'-broom, yellowing, reddening, plant sterility and decline (Figs. 1, 2). Total DNA was extracted from leaves and leaf petioles of five symptom-bearing and five symptomless C. virgatus plants using a CTAB-based method. DNA samples were tested for the presence of phytoplasmas by direct-PCR using primer pair P1/P7 (Lee et al., 1998) and nested PCR using primer pairs P1/P7 and R16F2n/R16R2 (Gundersen & Lee, 1996). The expected amplicons of c. 1,800 bp and 1,250 bp were obtained in direct and nested-PCR respectively, from all diseased plants but not symptomless ones. Two R16F2n/R16R2 amplicons were randomly selected and cloned into Escherichia coli DH5α cells using the PCR TA Cloning Kit (SinaClon, Cat. No.:CL5841, Iran) and three plasmid DNAs from recombinant colonies were sequenced. The obtained sequences were identical, and a consensus sequence was deposited in GenBank (Accession No. ON361565). BLASTn analysis of the obtained sequence showed the highest sequence identity (98.7-99.9 %) with phytoplasmas related to ‘Ca. P. aurantifolia’ (16SrII group). Phylogenetic analysis with MEGA11 software using the neighbour-joining method Fig. 3 revealed that the WBDCV phytoplasma clustered within the ‘Ca. P. aurantifolia’ (16SrII) group clade, closest to the strains of subgroup 16SrII-C. In Iran, phytoplasma strains belonging to 16SrII-C subgroup have been reported previously in Bushehr, Fars (adjacent to Hormozgan province) and Yazd provinces in several plant species including Daucus carota, Medicago sativa, Prunus dulcis, Vicia faba (Siampour et al., 2019), Alhagi maurorum and Artemisia sieberi (Esmaeilzadeh-Hosseini & Salehi, 2021); however, this is the first report of a ‘Ca. P. aurantifolia’-related strain in C. virgatus associated with WBDCV in Iran and most probably, in the world. The authors would like to thank the head of the Fars Agricultural and Natural Resources Research and Education Centre, Shiraz, Iran, for support and encouragement.

First report of a ‘Candidatus Phytoplasma australasia’‐related strain (16SrII‐D) associated with crown choking of areca palm in India

Areca palm (Areca catechu) is an economically important crop that is grown primarily in South Asia and South India, mainly in the states of Karnataka, Kerala and Maharashtra. With 0.73 million hectares cultivated and 1.2 million tonnes produced, India ranks first in world areca nut production. The state of Karnataka alone accounts 80% of annual production in India. During January 2021, crown choking symptoms (locally named “Hidimundige”), which resemble phytoplasma symptoms were observed on areca palm plantations. The highest disease incidence (50%) was recorded in Ajjarani, Sirsi Taluk and Uttara Kannda, followed by 30% in Haralakatta, Channagiri Taluk, Davanagere, Kelaginuru and Honnavara Taluk, in the district of Uttara Kannada (Karnataka state). The lowest disease incidence was 1% for the Dakashina Kannada and Tumkur districts in Karnataka.The most prominent symptom in infected palms was short, dark green leaves with a wavy leaf lamina. Leaflets failed to unfold properly leading to a bunchy top or witches’ broom appearance, and became crinkled and brittle. Reduced internodal length, drying of inflorescences and tapering of stem at the apex were also recorded. The roots became black, crinkled and brittle, and a pinkish or brownish discoloration was observed in the phloem. Affected plants failed to produce new leaves and existing leaves fell leading to a bare stem and death of the plant in quick succession (Figs. 1-3). About 80–100% of the plants died in affected areas. Seven leaf samples were collected from diseased palms growing in the Zonal Agricultural & Horticultural Research Station, Keladi Shivappa Nayaka University of Agricultural and Horticultural Sciences in Shivamogga, Karnataka (Latitude: 13° 55' 47.748“. Longitude: 75° 34' 5.1636”) during March 2022. Typical phytoplasma pleomorphic bodies lacking cell walls were seen in the sieve tube cells of leaf midrib sections from diseased areca palms, when examined by transmission electronic microscopy (Spurr, 1969) at the Sri Sai Histology Center, Hyderbad India (Fig. 4). The phytoplasma cell sizes ranged from 500 - 2000 nm. Seven leaf and three inflorescence samples from diseased palms and two leaf samples from asymptomatic palms were collected from the same location. Total DNA was extracted by the CTAB method and subjected to direct PCR using primers P1 (Deng & Hiruki, 1991) and P7 (Schneider et al., 1995) and nested PCR with R16F2n/R16R2 primers (Gundersen & Lee, 1996) that amplify the phytoplasma 16S rRNA gene. The expected direct (1.8 Kb) and nested (1.25 Kb) PCR products were obtained from all the diseased plant samples and positive reference controls but not from the symptomless plants. The R16F2n/R2 PCR products were cloned and sequenced directly at Biokart Pvt. Ltd., Karnataka, India, and the sequence deposited in GenBank (Accession No. ON606488). The R16F2n/R2 sequence was subjected to BLAST analysis and showed the highest nucleotide identity (99.92%) with 16SrII phytoplasma isolates, ‘Candidatus Phytoplasma aurantifolia’ (MT555412, MT555411) identified in Croton bonplandianus in Maharashtra, India. Virtual RFLP analysis of the R16F2n/R2 sequence with iPhyClassifier yielded an RFLP pattern identical to that of the 16Sr group II, subgroup D (Y10097) with a similarity coefficient of 1.00. Phylogenetic analysis (MEGA 7.0.18) using the neighbour-joining algorithm with selected phytoplasma reference strains confirmed that the areca palm phytoplasma clustered within the clade of phytoplasma strains of the 16SrII group (Fig. 5). In India, the cultivation of areca nut is mostly limited to the states of Assam, Karnataka and Kerala, and palms in these areas are affected by a yellowing disease named yellow leaf disease (YLD). YLD is one of the most important diseases of this palm species (Nambiar, 1949) and it is associated with the 16SrXI phytoplasma group, ‘Ca. P. oryzae’. (Chaithra et al., 2014). ‘Ca. P. aurantifolia’ is known to infect several plant species in India (Rao et al., 2017). However, this is the first report of a ‘Ca. P. aurantifolia’- related strain causing crown choking of Areca catechu in India. The impact of crown choking on areca nut yield, the geographic distribution of the disease and its epidemiological implication in India are currently under investigation.

Identification of 16SrII group phytoplasma strain associated with witches’ broom of spinach and hemp in India

Identification of 16SrII group phytoplasma strain associated with witches’ broom of spinach and hemp in India

Complete genome sequences of phytoplasma strains in group 16SrII associated with Parthenium phyllody in India

Complete genome sequences of phytoplasma strains in group 16SrII associated with Parthenium phyllody in India

First Report of 'Candidatus Phytoplasma asteris' (16SrI group) Associated with Murraya exotica Witches'-Broom Disease in Taiwan.

Murraya exotica L., commonly known as orange jasmine, is an evergreen shrub belonging to the Rutaceae family. It has long been used as traditional Chinese medicine for treating abdominal pain, toothache, scabies, and other disorders (Liu et al. 2018). M. exotica is widely grown as a garden bush in Taiwan. A prokaryotic pathogen, 'Candidatus Liberibacter asiaticus' (Damsteegt et al. 2010), reportedly could infect M. exotica, but there is no reported phytoplasma disease in M. exotica. In June 2020, M. exotica plants exhibiting witches'-broom (WB), leaf yellowing, and small leaves (Fig. s1) were observed in a horticultural landscaping field in Taichung City, Taiwan. It was estimated that more than 70% of M. exotica plants within a single area were affected. DNA was extracted separately from petioles of five symptomatic and one asymptomatic plants using a modified CTAB method (Echevarría-Machado et al. 2005) and used for nested PCR with two universal primers, P1 (Deng and Hiruki 1991)/P7 (Schneider et al. 1995) followed by R16F2n/R16R2 (Gundersen and Lee 1996) to amplify a 1.2-kb 16S rRNA fragment. PCR was also conducted by primers, rp(I)F1A/rp(I)R1A to amplify a partial ribosomal protein S3 and L22 (rplV-rpsC) fragment (Lee et al. 2004). Expected 1.2-kb bands were amplified from DNA extracted from all symptomatic plants, whereas no bands were amplified from that of the asymptomatic plant. The amplicons were cloned, sequenced with an ABI 3730 automatic sequencer (Applied Biosystems, Hammonton, NJ, USA) in Biotechnology Centre DNA-sequencing facility at National Chung Hsing University (NCHU) and deposited in GenBank. BLAST analysis revealed that 16S rDNA sequences (MZ373297 and MZ373298) shared 100% identity to each other and both shared 99.4% identity with those of several phytoplasma strains, e.g., rapeseed phyllody phytoplasma (CP055264), Brassica sp. phyllody phytoplasma (MN877914), Plumbago auriculata leaf yellowing phytoplasma (MN239504), and aster yellows phytoplasma (MK992774), which all belonging to the 16SrI group, by using the CLUSTAL W Methods of MegAlign program (DNASTAR, Inc., Madison, WI, USA). Further analysis using iPhyClassifier tool (https://plantpathology.ba.ars.usda.gov) indicated that the virtual restriction fragment length polymorphism (RFLP) patterns derived from the 16S rDNA F2nR2 fragment of the M. exotica WB phytoplasma was most similar to the reference pattern of the 16SrI-B subgroup, with a pattern similarity coefficient of 0.97 and shared 99.3% sequence identity to 'Candidatus Phytoplasma asteris' (M30790). The partial rplV-rpsC gene sequence (OM275408) showed 99.7% of sequence identities to those of rapeseed phyllody phytoplasma (CP055264), plum witches'-broom phytoplasma (MH061366) and oilseed rape phytoplasma (KX551965), by using the CLUSTAL W Methods of MegAlign program. Taken together, we concluded that the phytoplasma strain associated with M. exotica WB disease was a strain belonging to a 16SrI. To the best of our knowledge, this is the first report of M. exotica being infected by a phytoplasma in the aster yellows group, and M. exotica may also serve as an intermediate reservoir host to other plants, e.g., wax apple, periwinkle and roselle, of 16SrI phytoplasma.

Development of a Specific Nested PCR Assay for the Detection of 16SrI Group Phytoplasmas Associated with Sisal Purple Leafroll Disease in Sisal Plants and Mealybugs.

Sisal purple leafroll disease (SPLD) is currently the most destructive disease affecting sisal in China, yet its aetiology remains unclear. In our previous research, it was verified to be associated with phytoplasmas, and nested PCR based on the 16S rRNA gene using universal primers R16mF2/R16mR1 followed by R16F2n/R16R2 was confirmed as the most effective molecular method for the detection of phytoplasmas associated with SPLD (SPLDaP). However, the method has a shortcoming of inaccuracy, for it could produce false positive results. To further manage the disease, accurate detection is needed. In this study, we developed a specific nested PCR assay using universal primers R16F2n/R16R2, followed by a set of primers designed on 16Sr gene sequences amplified from SPLDaP, nontarget bacteria from sisal plants, and other phytoplasma subgroups or groups. This established method is accurate, specific, and effective for detection of 16SrI group phytoplasma in sisal, and its sensitivity is up to 10 fg/μL of total DNA. It also minimized the false positive problem of nested PCR using universal primers R16mF2/R16mR1 followed by R16F2n/R16R2. This method was further used to verify the presence of phytoplasma in Dysmicoccusneobrevipes, and the results showed that D. neobrevipes could be infected by SPLDaP and thus could be a candidate for vector transmission assays.

Association of phytoplasmas with a new disease of potato crops in cundinamarca, Colombia

Cundinamarca is the major potato producer region in Colombia. Potato growers first reported the presence of new symptoms affecting commercial potato fields in Cundinamarca as far back as 2012. These included foliage yellowing, curling on the upper leaf side and purple margins, occasionally with purple coloration of the underside veins, excessive shoot proliferation, abnormally short or long internodes and abnormal leaf development. Plants had a distorted pattern of growth suggesting a possible phytoplasma cause. Phytoplasmas were detected by nested PCR of the phytoplasma 16S rRNA gene and we confirmed the presence of ‘Candidatus Phytoplasma asteris’ (group 16SrI) and ‘Candidatus Phytoplasma fraxini’ (16SrVII) by RFLP and sequencing of samples from commercial plots. Of 152 symptomatic and non-symptomatic potato plants tested, 33.5% were positive for one of the identified phytoplasmas or both (mixed infections). Phytoplasmas were also observed by electron microscopy in symptomatic plants. Both phytoplasmas were transmitted in tubers produced by infected plants and by the insect vector Exitianus atratus (Hemiptera: Cicadellidae) to healthy test potato plants. Daughter plants from infected tubers and the healthy plants inoculated via the insect vector developed the same symptoms observed in field grown potato plants, in single and mixed infections of both phytoplasmas. Our results confirm the presence of a new disease of potato in Cundinamarca, Colombia, which we named yellowing and rolling-leaf phytoplasma (fitoplasma del amarillamiento y enrrollamiento de hojas).

‘Candidatus Phytoplasma solani’ (Subgroup 16SrXII-A) Associated with Nicotiana tabacum Leaf Abnormality in Turkey

Tobacco (Nicotiana tabacum) is among the agricultural products with the highest added value in Turkey. Although frequently associated with its negative effects on human health, it also provides important contributions to the Turkish economy with the employment it creates in rural areas and continues to be a strategic product. Many postgraduate theses and studies related to the sociological and economic importance of the production of this plant, which is of great importance for our country, have been carried out. However, there are very limited studies on plant diseases in tobacco production areas in Turkey. Phytoplasma is one of the important plant pathogens that cause yield loss in tobacco. Since available data on phytoplasma diseases on tobacco was very scarce worldwide, field surveys to collect samples showing phytoplasma infection-like symptoms such as yellowish color changes, leaf blisters, proliferation, dwarfism, and other physical abnormalities were carried out in Çanakkale and Balıkesir provinces of Turkey from June to August 2021. The presence of phytoplasmas in six samples was confirmed by 16S ribosomal DNA amplification by nested-PCR using universal phytoplasma primer sets, which also suggested the pathogen associated with the symptoms on tobacco. According to phylogenetic study and virtual-RFLP analysis using AluI and MseI endonuclease enzymes, the six Turkish tobacco phytoplasma strains all belong to group 16SrXII and have more than 99% nucleotide sequence identity with some members of ‘Candidatus Phytoplasma solani’ of the taxonomic subgroup ‘stolbur’ (16SrXII-A). Genetic distances analysis indicated that group 16SrI was more closely related to 16SrXII than 16SrVI, in agreement with the groups clustering in the phylogenetic tree. Neutrality tests found that 16SrI and 16SrXII groups are experiencing expanding or bottleneck selections, probably due to new mutations in the 16S rRNA gene fragment. Meanwhile, 16SrVI populations are shown to be undergoing balancing selections, indicating that its isolates have evolved for a long time.

Genetic diversity of phytoplasmas associated with several bamboo species in India

AbstractSymptoms of phytoplasma diseases, including witches' brooms, shoot proliferation, little leaf, yellowing and decline were observed in eleven species of bamboos in eight states of India. Symptomatic bamboo samples were indexed for phytoplasma presence using universal phytoplasma‐specific 16S rRNA gene primer pairs. Sequence comparison analysis and virtual RFLP analysis of 16S rRNA sequences indicated that the symptomatic bamboo samples of Dendrocalamus asper, Dendrocalamus strictus and Pseudoxytenanthera stocksii from Tripura, Uttar Pradesh, Uttarakhand and New Delhi and Bambusa bambos, Bambusa nutans, Bambusa pallida,Bambusa tulda,Dendrocalamus hamiltonia and D. strictus from Karnataka were infected with ‘Candidatus Phytoplasma australasia’‐related strains (16SrII‐C or 16SrII‐D); Bambusa vulgaris,D. asper and P. stocksii from Karnataka were positive for ‘Ca. P. asteris’ strains (16SrI‐B), and one phytoplasma in B. nutans from Sikkim was identified as ‘Ca. P. cynodontis’ strain (16SrXIV‐A). Weeds and leafhoppers from the vicinity of bamboo plantations were also tested, and 16SrII group of phytoplasmas was identified in eight symptomatic weed species (Ageratum conyzoides,Cannabis sativa,Cleome viscosa,Datura stramonium, Parthenium hysterophorus,Ocimum canum, Phyllanthus niruri and Tephrosia purpurea) from five different states and one leafhopper species (Mukaria spledida) from three states. These results suggest that wide genetic diversity of phytoplasma exists in various bamboo species in different states of India. The report of 16SrI‐B, 16SrII‐D and 16SrXIV‐A subgroups of phytoplasmas infecting eight bamboo species except D. strictus are the first records in the world.

First report of a ‘Candidatus Phytoplasma australasia’ (16SrII‐D) strain associated with witches’ broom and little leaf symptoms of Martynia annua in India

Martynia annua (Martyniaceae) is a small, herbaceous annual plant distributed across India. It is used for the treatment of epilepsy, tuberculosis, sore throats and wounds (Kaushik et al., 2021). In August 2021, symptoms of witches’ broom and little leaf were observed in M. annua in the Anantapur forest area, Andhra Pradesh. India. Leaves were collected from two diseased and two symptomless plants (Figures 1 and 2). Total DNA was extracted from 100 mg of leaf midribs using a modified CTAB DNA extraction method (Murray & Thompson, 1980). DNA extracts were analysed by direct and nested PCR with universal phytoplasma 16S rRNA primers, P1/P7 (Deng & Hiruki, 1991) and R16F2n/R16R2 (Gundersen & Lee, 1996), respectively. Amplicons of the expected size (c. 1.8 kb after direct PCR and c. 1.3 kb after nested PCR) were obtained from diseased samples but not symptomless plants. Nested PCR R16F2n/R16R2 products were directly sequenced (Eurofins Genomics India Pvt., Ltd., India) and the consensus sequence was deposited in GenBank (Accession No. OK135941). A BLASTn analysis revealed that the M. annua phytoplasma 16Sr DNA sequence shared 100% sequence identity with those of 16SrII group phytoplasmas, including ‘Candidatus Phytoplasma australasia’ (MN565885, MN565886) and ‘Ca. Phytoplasma aurantifolia’ (MK217101, MK421430). Phylogenetic analysis (Tamura et al., 2007) based on the R16F2n/R2 sequence and performed with the neighbour-joining method (MEGA software version 7.1) revealed that the Anantapur 16SrII phytoplasma had the closest relationship with 16SrII, subgroup D isolates, particularly with the strain ‘Ca. P. australasia’ (Y10096) (Figure 3). Virtual RFLP profiles using restriction enzyme AluI and the R16F2nR2 sequence were generated with iPhyClassifier (https://plantpathology.ba.ars.usda.gov/cgi-bin/resource/iphyclassifier.cgi). The RFLP patterns of the Anantapur phytoplasma 16S rDNA F2nR2 sequence with AluI enzyme agreed with the phylogenetic grouping. The Anantapur phytoplasma was confirmed as a member of the 16Sr group II, subgroup D with a similarity coefficient of 1.00, compared with virtual RFLP profiles of the ‘Ca. P. australasia’ strain (Y10097). This is the first report of a ‘Ca. Phytoplasma australasia’ strain (16SrII-D) associated with witches’ broom and little leaf symptoms of M. annua in India. Since the 16SrII group has a wide host range and occurrence in India, and particularly the subgroup 16SrII-D in the south region, the present finding has a significant impact for future epidemiological studies. The authors would like to thank the support and encouragement from the Associate Director of the Research, Regional Agricultural Research Station, Tirupati.

First report of a 16SrII‐V phytoplasma associated with peanut phyllody in India

Peanut (Arachis hypogaea, Fabaceae), also known as groundnut, is an important oilseed and food legume crop cultivated in a wide range of climatic conditions across the globe. India is the second largest producer of peanut in the world after China (Food and Agriculture Organization of the United Nations, 2022). Peanut plants exhibiting phyllody, little leaf, excessive shoot proliferation, negative geotropism of pegs with absence of pod formation were observed in February 2021 in Pune, Maharashtra, India (Figures 1, 2). The diseased plants could be easily identified in the field due to their distinct appearance, suggestive of the presence of phytoplasma. Eight peanut fields were surveyed and the overall average disease incidence was four percent. Ten diseased and four asymptomatic plants were sampled to identify and characterise the associated phytoplasma. DNA was extracted from leaf petioles (100 mg) using DNeasy® Plant Mini Kit (Qiagen, Germany). The extracted DNA was subjected to PCR using P1/P7 universal primers (Deng & Hiruki, 1991; Schneider et al., 1995). Products from the first round PCR were used in nested PCR with R16F2n/R2 primers (Gundersen & Lee, 1996). Amplicons of the expected size, c. 1.8 kb and 1.2 kb were obtained in all diseased samples in PCR and nested PCR assay, respectively. No amplification was recorded in asymptomatic samples. The sequences of the nested PCR products were identical to each other and the sequence was deposited in GenBank (Accession No. ON139214). A BLASTn search showed more than 99% sequence identity with phytoplasma sequences including peanut witches’-broom (JN681274; L33765), Desmodium ovalifolium witches’-broom (MK956144), soybean witches’-broom (MW115943) and cowpea phyllody (MZ831318). The peanut phyllody phytoplasma sequence clustered with selected sequences belonging to the 16SrII phytoplasma group following phylogenetic analysis using the neighbour-joining method (MEGA X program) (Figure 3). Virtual RFLP using iPhyClassifier (https://plantpathology.ba.ars.usda.gov/) demonstrated that the peanut phyllody phytoplasma is affiliated to group 16SrII, subgroup V. Phytoplasmas belonging to subgroup 16SrII-V have been recently reported to infect legumes such as mungbean and soybean in Taiwan (Chen et al., 2021; Wang et al., 2021). To our knowledge, this the first report of a 16SrII-V phytoplasma associated with phyllody in peanut as well as the first report of the occurrence of a 16SrII-V phytoplasma in India. The finding is of epidemiological importance as the phytoplasma infection reduces pod formation leading to yield and economic losses for farmers. Moreover, infected peanut plants could serve as natural reservoirs of the phytoplasma aiding its spread to other legume, vegetable and flower crops cultivated in the vicinity. The authors thank the Director, ICAR-IARI, New Delhi and Head, ICAR-IARI, RS, Pune for providing lab facilities.

Effect of Phytoplasma Associated with Sesame Phyllody on Ultrastructural Modification, Physio-Biochemical Traits, Productivity and Oil Quality.

Phytoplasmas are obligate cell-wall-less plant pathogenic bacteria that infect many economically important crops, causing considerable yield losses worldwide. Very little information is known about phytoplasma–host plant interaction mechanisms and their influence on sesame yield and oil quality. Therefore, our aim was to explore the ultrastructural and agro-physio-biochemical responses of sesame plants and their effects on sesame productivity and oil quality in response to phytoplasma infection. Sesame leaf samples exhibiting phyllody symptoms were collected from three experimental fields during the 2021 growing season. Phytoplasma was successfully detected by nested- polymerase chain reaction (PCR) assays using the universal primer pairs P1/P7 and R16F2n/R16R2, and the product of approximately 1200 bp was amplified. The amplified product of 16S rRNA was sequenced and compared with other available phytoplasma’s 16S rRNA in the GenBank database. Phylogenetic analysis revealed that our Egyptian isolate under accession number MW945416 is closely related to the 16SrII group and showed close (99.7%) identity with MH011394 and L33765.1, which were isolated from Egypt and the USA, respectively. The microscopic examination of phytoplasma-infected plants revealed an observable deterioration in tissue and cell ultrastructure. The primary and secondary metabolites considerably increased in infected plants compared with healthy ones. Moreover, phytoplasma-infected plants showed drastically reduced water content, chlorophyll content, growth, and yield components, resulting in 37.9% and 42.5% reductions in seed and oil yield, respectively. The peroxide value of the infected plant’s oil was 43.2% higher than that of healthy ones, suggesting a short shelf-life. Our findings will provide a better understanding of the phyllody disease pathosystem, helping us to develop effective strategies for overcoming such diseases.

Identification of ‘Candidatus Phytoplasma aurantifolia’ in pigeon pea with little leaf disease in Karnataka, India

Pigeon pea is an important pulse crop grown in tropical regions and mainly cultivated in semi-arid regions of India. Phytoplasma little leaf symptoms were observed on BGR-1 variety of pigeon pea in an experimental plot in Karnataka. The affected leaf samples were subjected to nucleic acid extraction for PCR amplification of phytoplasma 16S rDNA by nested PCR. The obtained product was sequenced and compared with the phytoplasma sequences available in the NCBI database. This sequence resulted having 100% nucleotide identity with a phytoplasma associated with sun hemp witches’ broom from Tirupati (GenBank accession number OL891522) placed in 16SrII group and with ‘Candidatus Phytoplasma aurantifolia’ that results therefore reported for the fist time in Karnataka state of India.

Presence and seed transmission of phytoplasmas in tomato fields in Italy

Tomatoes are the second most consumed vegetable, quite often severely affected by phytoplasma-associated diseases worldwide. The identification and confirmation that the phytoplasmas associated with the disease in Southern Italy are also transmitted through seeds was achieved. DNA extractions from mother plants collected in 2020 and 2021 and the seedlings from these plants, tested at different times, allowed the detection of phytoplasmas in both mother plants and seedlings grown under an insect-proof greenhouse. Phytoplasmas enclosed in groups 16SrX-B (‘Candidatus Phytoplasma prunorum’) and 16SrV-A (‘Ca. P. ulmi’) were identified in 2020 samples, and the same phytoplasma groups were identified in the seedlings. Phytoplasmas enclosed in groups 16SrI and 16SrXII were identified in mother plants collected in 2021 and were also identified in the seedlings. Moreover, a difference in germination percentage was observed. The 16SrXII-A group phytoplasmas detected in the mother plants and were identified as variants St5 for the stamp gene, tuf-b1 for the tuf gene and Vm44 for the vmp1 gene. This research is a significant confirmation of previous laboratory results on transmission of phytoplasmas through seeds from several diseased plant species. The epidemiological role of phytoplasma seed transmission is an essential knowledge for efficient management strategy planning for a seed production aimed at reducing the impact of phytoplasma-associated diseases in horticultural relevant crops such as tomatoes.

Acerola shoot proliferation induced by a phytoplasma enclosed in the subgroup 16SrIII-F

Acerola bushes were observed showing symptoms of shoot proliferation, generalized stunting, yellowing and decline. Since these symptoms are typically induced by phytoplasmas, this survey was carried out with the aim of detecting, identifying and classifying the supposed phytoplasma present in symptomatic bushes. Total DNA was extracted from symptomatic and asymptomatic samples and used in nested PCR conducted by the primer pairs R16mF2/mR1 followed by R16F2n/R2. Amplifications of expected genomic fragments of 1.2 kb revealed the presence of phytoplasma in 73 % of the symptomatic samples. Molecular analyses, using computer-simulated RFLP patterns, similarity coefficient calculation and phylogenetic analysis allowed for classifying the bacterium as a ‘Candidatus Phytoplasma pruni’ – related strain (subgroup 16SrIII-F). The phytoplasma induced the same symptoms in healthy acerola plants inoculated by grafting and showed molecular identity with the strain identified in naturally infected bushes. Although various strains belonging to distinct subgroups within the 16SrIII group have been previously identified in Brazil, this is the first report of the presence of a representative of the 16SrIII-F subgroup in the Brazilian agroecosystem. Considering that phytoplasmas can be systemically distributed throughout the plant and acerola plants are vegetatively propagated, it is recommended that propagation material be obtained from mother plants free of the pathogen.