First Report of 'Candidatus Phytoplasma citri' related strain (16SrII-C subgroup) associated with phyllody disease of cumin in India.

Cumin (Cuminum cyminum L.), is an important export-oriented seed spice crop for India. Cumin is popularly used for flavouring food, including soups, pickles and vegetables, and for herbal medicine. India is the largest producer, consumer and exporter of cumin seed with an annual production of 0.795 million tones over an area of 1.09 million hectares. During 2020-21, India exported about 0.299 million tons of cumin worth of Rs 33280 million (Anonymous, 2021). Recently, phytoplasma suspected symptoms were observed in cumin at Agricultural Research Station, Mandor, Jodhpur, Rajasthan, India from 2019. The symptoms related to phytoplasma infection were first recorded after 70-75 days of sowing in the month of January of the years 2019 to 2022. The major symptoms recorded were yellowing, phyllody, witches-broom, yellowing and deformed elongated seeds. Disease incidence was recorded as 0.25-1.0%, 0.5-1.5%, 0.5-2.5 % and 0.5-10.6% during the years 2019, 2020, 2021 and 2022, respectively using quadrate method. In 2022, among different genotypes assessed GC 4, MCU 73, MCU 105, and MCU 32 exhibited lower disease incidences ranging from 0.5% to 1.5%, while MCU 78 recorded the highest disease incidence at 10.6%. To detect the association of phytoplasma with symptomatic cumin samples, genomic DNA was extracted from four representative cumin genotypes (CuPP-MND-01 to CuPP-MND-04) and one asymptomatic cumin plant using the Qiagen DNeasy plant mini kit (Germany). The extracted DNA was amplified using nested PCR assays with universal phytoplasma detection primers for 16S rRNA gene (P1/P7 and R16F2n/R16R2) (Schneider et al., 1995; Gundersen and Lee, 1996) and secA gene specific primers (SecAfor1/SecArev3 followed by nested PCR primers SecAfor5/ SecArev2) (Hodgetts et al. 2008; Bekele et al. 2011). The amplicons of ∼1.25 kb with 16S rRNA gene and ∼600 bp with secA gene specific primers were amplified in all symptomatic cumin plant samples and positive control of brinjal little leaf. PCR amplified products from the four selected positive samples (CuPP-MND-01 to CuPP-MND-04) of 16S rRNA gene and secA gene, were sequenced from both ends. The 1,245 bp sequences were deposited in GenBank (OQ299007-10), which showed 100% sequence identity with each other and 99.4% identity with 'Candidatus Phytoplasma citri' reference strain (GenBank accession: U15442) (Rodrigues Jardim et al. 2023). The phylogenetic analysis and virtual RFLP analysis using 17 restriction enzymes of 16S rRNA gene sequences through iPhyclassifier allowed affiliating the cumin phytoplasma strains with 16SrII-C subgroup strain with a similarity coefficient of 1 to the reference pattern of 16Sr group II, subgroup C (GenBank accession: AJ293216) (Zhao et al. 2009). In addition, the phylogenetic analysis of the secA gene-based sequences (OQ305073-76) further confirmed the close association of 16SrII-C group phytoplasmas with phyllody and witches' broom disease of cumin. Earlier 16SrII-C subgroup phytoplasma has been reported from various crops and weeds in India (Rao et al. 2021). However, no phytoplasma association has been reported earlier with cumin in India and abroad. To the best of our knowledge, this is the first report on the association of 16SrII-C group phytoplasma causing phyllody, witches' broom in cumin genotypes. This report has economic and epidemiological implications and needs immediate attention to reduce export losses due to phytoplasma disease in cumin and to prevent the potential spread to other crops.

First Report of Candidatus Phytoplasma asteris (16SrI-B subgroup) associated with phyllody disease of fenugreek in the world.

Fenugreek (Trigonella foenum-graecum) is a leafy vegetable and spice crop, native to Indian subcontinent and Eastern Mediterranean region. Phytoplasma infection symptoms were observed in fenugreek at ICAR-National Bureau of Plant Genetic Resources Regional Station, Jodhpur and Agricultural Research Station Mandore Jodhpur, Rajasthan, India. The first appearance of phytoplasma suspected symptoms of little leaf was recorded after 50 days of sowing in the months of January 2022. The major symptoms recorded were virescence, phyllody, shoot proliferation, witches-broom, little leaf, yellowing and overall stunted growth in 146 germplasm accessions at NBPGR research farm, Jodhpur and one major commercially cultivated variety RMT 305 at Mandore Jodhpur. Ten samples from symptomatic and five samples from asymptomatic fenugreek plants were collected and processed for total DNA extraction using the Qiagen DNeasy plant mini kit (Germany). The extracted DNA was amplified using nested PCR assays with universal phytoplasma detection primers for 16S rRNA gene (P1/P7 and R16F2n/R16R2) and secA gene specific primers (SecAfor1/SecArev3 and SecAfor2/SecArev3) (Schneider et al. 1995; Gundersen and Lee 1996; Hodgetts et al. 2008). The amplicons of ∼1.25 kb with 16S rRNA and ∼480 bp with secA gene specific primers were amplified in all symptomatic fenugreek samples. In negative control (asymptomatic plants) no amplification was observed with either of gene specific primers in gel electrophoresis. PCR amplified products from the six selected positive samples (FPP-NBPGR-J-01 to FPP-NBPGR-J-04 and FPP-MND-01 to FPP-MND-02) of 16S rRNA and secA gene, were sequenced from both ends. Sequences were deposited in the NCBI GenBank with accession numbers ON756108-ON756113 for 16S rRNA gene sequences and ON745809 to ON745814 for secA gene sequences. BLAST analysis of 16S rRNA and secA sequences revealed 100% sequence identity among themselves and 99.95 to 100% sequence identity with the earlier reported phytoplasma strains of aster yellows group related phytoplasma strains (GenBank Acc. No. MN239504, MN080270) belonging to Ca. P. asteris (16SrI group). Further analyses of the 16S rRNA and secA gene-based phylogenetic tree and the iPhyClassifier-based virtual RFLP analysis of 16S rRNA gene study demonstrated that the phytoplasma associated with fenugreek phyllody belonged to 16Sr group I ('Ca. P. asteris') and subgroup B (GenBank accession AP006628), with similarity coefficient of 1.0. Earlier association of 16Sr-II-D subgroup (Ca. P. australasiae) with fenugreek as host was reported from Pakistan (Malik et al., 2020). To the best of our knowledge, this is the first report of a 'Ca. P. asteris', 16SrI-B subgroup related phytoplasma strain associated with fenugreek phyllody in the world. The 16SrI-B phytoplasma strain is a widely distributed strain associated with several agricultural and horticultural crops of India (Rao 2021). This is not only the first instance of fenugreek phyllody disease found in India, but also the first instance of fenugreek phyllody caused by 16SrI-B subgroup phytoplasma worldwide. This report has epidemiological significance and needs immediate attention, as fenugreek is one of the most common seed spice crop being grown all over India.

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.

First report of Candidatus Phytoplasma cynodontis (16SrXIV-A subgroup) associated with cauliflower phyllody and flat stem in India.

Symptoms of suspected phytoplasma infection were observed in cauliflower (Brassica oleracea var. botrytis) (cultivar NS60N) at Integrated Farming System Research Station, Trivandrum, Kerala, India (08o28'28"N, 76o57'47"E) in April-2021. The disease incidence was recorded up to 10% in different fields. The disease manifested as stunting, phyllody, floral malformation and flattening of stem (Fig.1A,B). Ten symptomatic and five asymptomatic plants were assayed for the presence of phytoplasma using nested PCR assays performed with P1/P7 and R16F2n/R16R2 primer pairs for 16S rRNA gene and SecAfor1/ SecArev3 and SecAfor2/ SecArev3 for secA gene (Deng and Hiruki 1991; Gundersen and Lee 1996; Hodgetts et al. 2008). The expected amplicons of ~1.25 kb and ~480 bp were consistently amplified in all the symptomatic cauliflower samples with the phytoplasma specific universal 16S rRNA and secA gene specific primers. Nested PCR products (~1.2 kb and 480 bp) amplified from cauliflower was cloned in EcoRI restriction sites of pGEM-T Easy vector (Promega, USA). The cloned nested PCR products were directly sequenced (16S rRNA gene: Acc. Nos. MZ196223, MZ196224; secA gene: MZ215721, MZ215722) in both forward and reverse directions which showed 99.77% sequence identity with Candidatus Phytoplasma cynodontis reference strain (Acc. No. AJ550984). Further analyses of the 16S rRNA and secA genes based phylogenetic tree (Fig. 2A and B) and the iPhyClassifier-based virtual RFLP analysis of 16Sr RNA gene study demonstrated that the phytoplasma-associated with cauliflower phyllody & flat stem disease (CaPP) belonged to 16SrXIV-A subgroup with a similarity coefficient of 1.0. No amplicon was observed from any of the asymptomatic cauliflower plants with the specific tested primers of both the genes. Earlier association of 16SrXV-A subgroup (Candidatus Phytoplasma brasiliense) and 16Sr III-J subgroup in Brazil (Canale and Badendo, 2013; Rappussi et al. 2012), 16SrII-A (Candidatus Phytoplasma aurantifolia) subgroup in China (Cai et al. 2016) and 16SrVI-A (Candidatus Phytoplasma trifolii) subgroup in Iran (Salehi 2007) were reported in cauliflower. Another species of cabbage, Brassica oleracea var. capitata L. was reported as host of Ca. P. trifloii (16Sr VI-D subgroup) from north India (Gopala et al. 2018). To our knowledge, this is the first report of a 'Candidatus Phytoplasma cynodontis', 16SrXIV-A subgroup related phytoplasma strain associated with cauliflower phyllody and flat stem in the world. The results described in this report confirm that the 16SrXIV-A phytoplasma, a widely distributed strain associated with sugarcane, wheat, grasses, sapota and many ornamentals in India (Rao 2021), has also infected cauliflower. This is not only the first instance of cauliflower phyllody disease found in India, but also the first instance of CaPP disease caused by 16SrXIV-A subgroup phytoplasma worldwide. This report has epidemiological significance and needs immediate attention, as cauliflower is the one of the most common vegetable crop grown all over India.

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

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

Identification and multilocus gene characterization of phytoplasmas associated with sweet cherry in India.

Symptoms of leaf roll, swollen nodes, flat branch and witches' broom were observed in five cultivars of sweet cherry from Srinagar, Jammu and Kashmir province, India, during 2019-2021. Phytoplasmas association were confirmed by amplifying 16S rRNA, secA, rp, tuf and secY genes with phytoplasma-specific primers in all symptomatic sweet cherry cultivars in nested PCR assays. Pairwise sequence comparison, phylogeny and virtual RFLP (16S rRNA gene) analyses confirmed the presence of 'Candidatus Phytoplasma asteris' and 'Ca. P. trifolii' strains in the sweet cherry samples. The incidence of flat branch and witches' broom symptoms associated with 'Ca. P. trifolii' varied from 5.8 to 25% in cultivars Bigarreau Nepoleon (Double), Bigarreau Noir Grossa and CITH-Cherry-9. However, incidence of leaf rolling, swollen nodes and bud proliferation associated with 'Ca. P. asteris' was recorded 7.5% in cultivar Stella and 10% in Sunburst, respectively, in the surveyed area. The multigene characterization of sweet cherry phytoplasma strains confirmed the validity of these molecular markers for identification of phytoplasmas enclosed in 16SrI and 16SrVI groups. The presence of phytoplasmas in sweet cherry is the first report from India.

High-Throughput RNA Sequencing of Mosaic Infected and Non-Infected Apple (Malus × domestica Borkh.) Cultivars: From Detection to the Reconstruction of Whole Genome of Viruses and Viroid.

Many viruses have been found associated with apple mosaic disease in different parts of the world. In order to reveal and characterize the viruses and viroids in symptomatic apple plants, next-generation sequencing (RNA seq.) of rRNA-depleted total RNA using Illumina Hiseq2500 was applied to two cultivars, Oregon Spur and Golden Delicious, with symptoms of mosaic and necrosis and one cultivar, Red Fuji, which was asymptomatic. The RNA sequencing detected five viruses, viz., apple necrotic mosaic virus (ApNMV), apple mosaic virus (ApMV), apple stem grooving virus (ASGV) and apple stem pitting virus (ASPV), apple chlorotic leaf spot virus (ACLSV), and one viroid i.e., apple hammerhead viroid (AHVd). RT-PCR amplification and sequencing also confirmed the presence of all these five viruses and viroids detected in HTS of total RNA. The complete genomes of five viruses and AHVd were reconstructed. The phylogenetic analysis of these viruses and AHVd revealed genetic diversity by forming subclusters with isolates from other countries. Recombination events were observed in all five viruses while single-nucleotide variants were detected only in ApMV and ApNMV. The absence of ApMV and ApNMV in asymptomatic samples from the same cultivars in an RT-PCR assay indicated that these two viruses are associated with mosaic disease of apples in India. This is the first viral genome analysis of symptomatic and asymptomatic apple plants and the first report of genome characterization of viruses associated with apple mosaic disease from India. High-throughput RNA sequencing is a powerful tool to characterize the genome of viruses and viroids in plants previously undetected by conventional methods. This would also help in the indexing and certification of large-scale germplasm.

Association of Apple necrotic mosaic virus (ApNMV) with mosaic disease in commercially grown cultivars of apple (Malus domestica Borkh) in India.

During the course of survey, an incidence of 7.14-90% of apple mosaic disease (AMD) was recorded in apple orchards in Jammu and Kashmir among various commercially grown cultivars. The maximum incidence of mosaic disease was observed in cultivar Golden Delicious. In addition to mosaic, symptoms of chlorosis, necrosis and ring spots were also observed. In the present study association of Apple necrotic mosaic virus (ApNMV) was confirmed by RT-PCR and sequencing of whole coat protein gene in samples tested negative for Apple mosaic virus (ApMV) in DAS-ELISA. Out of 18 samples tested in RT-PCR, ten were found positive for ApNMV. Out of ten ApNMV positive samples, amplicon of 680 bp of samples representing five cultivars were sequenced and sequence analysis showed 89-91% sequence identity with ApNMV. The phylogenetic analysis grouped Indian isolates into two sub-clusters under one major cluster (ApNMV group). The sub-cluster-I, included ApNMV isolates from cultivars, Oregon Spur, Red Delicious and Fuji Aztec along with Chinese and Korean isolates. Sub-cluster-II included ApNMV isolates associated with Golden Delicious and Royal Delicious. The comparison of coat protein gene-based sequence identity matrix showed maximum and minimum similarity of 89-99% with ApNMV isolates from China. It also showed maximum identity with PNRSV (61.6%) and ApMV (52.8%) under subgroup 3 of genus Illarvirus. Our study indicates that the ApNMV is commonly associated with AMD in India and may be a major cause of the mosaic disease in apple cultivars. To the best of our knowledge, this is the first report of the association of ApNMV with apple mosaic disease from India.