Association Of Phytoplasma Research Articles

First report of 'Candidatus Phytoplasma trifolii' related strain causing witches’ broom disease of black cumin (Nigella sativa L.) in India

In spring, 2024, black cumin plants in Uttar Pradesh, India, exhibited leaf yellowing and witches' broom symptoms. Phytoplasma association was confirmed by PCR assays using universal primers for the 16S rRNA and secY genes. BLASTn, phylogenetic analysis and Virtual RFLP analysis confirmed the association of 'Candidatus Phytoplasma trifolii' (16SrVI-D) subgroup related strain with the Nigella sativa plants, marking as the first report worldwide.

Multilocus sequence analysis of ‘Candidatus Phytoplasma asteris’ associated with phyllody of cucumber in India and development of loop‐mediated isothermal amplification (LAMP) assay for its detection

A survey assessed cucumber phyllody disease incidence in Chikkaballapura, Bengaluru Rural, Bengaluru Urban, and Bagalkote districts of Karnataka, India. The average disease incidence ranged from 8 to 18 %. A total of 9 samples showing phyllody and little leaf symptoms were collected and association of phytoplasma was confirmed through 16S rRNA gene amplification, using universal primer pair P1/P7, and followed by nested PCR using primer R16F2n/R16R2. Further, characterization through multilocus sequence analysis targeting secY and rpl22 genes confirmed all nine strains as part of the 16SrI group ('Candidatus Phytoplasma asteris'). Additionally, four weed species (Lecusa aspera, Parthenium hysterophorus, Acaranthus sp., and Amaranthus viridis) found in cucumber fields tested positive for phytoplasma (16SrI) disease. In-silico RFLP analysis of the amplified F2n/R2 region of the 16S rRNA gene indicated that two phytoplasma strains (CuPP1 and CuPP3) from Chikkaballapura belonged to subgroup X (16SrI-X), one phytoplasma strain (CuPP2) belonged to subgroup B (16SrI–B). While the remaining strains from Bangalore Urban, Bengaluru Rural, and Bagalkote districts belonged to subgroup B (16SrI–B). A loop-mediated isothermal amplification (LAMP) detection protocol was also developed for 'Ca P. asteris', targeting the phytoplasma 16S ribosomal DNA. Comparison of assay sensitives demonstrated that LAMP technique is highly efficient and could detect the presence of phytoplasma up to 50 fg of template DNA. LAMP-based detection offers superior ease of use, cost-effectiveness, and rapid results. To the best of our knowledge, this is the first report of phytoplasma causing phyllody disease in cucumber crop in India.

First Report of a 'Candidatus Phytoplasma sacchari'-Related Strain Associated with Yellowing and Decline of Silver Bluestem in Texas, U.S.A.

Silver bluestem [Bothriochloa laguroides (DC.) Herter] is a warm-season grass native to Texas. This perennial grass plays a crucial role in maintaining ecological balance and supporting wildlife in the region. In September 2022, while investigating the ecological impact of invasive grass species on a grassland located near Pipe Creek (TX), B. laguroides plants were observed showing symptoms that included yellowing of the blades and occasionally brown discoloration of the midveins and stems (Fig. S1). Disease incidence was estimated as 2% of silver bluestem plants in the 2 hectares surveyed. To investigate the possibility of a phytoplasma association with the symptoms, four symptomatic and four asymptomatic leaf samples were collected for further study. Total DNA was extracted from leaf midribs using a DNeasy Plant Mini Kit (Qiagen). The DNA extracts were tested using a phytoplasma-specific quantitative PCR assay (Hodgetts et al. 2009), which identified two out of the four symptomatic B. laguroides samples as positive for phytoplasmas. A semi-nested PCR assay for amplification of the 16S rRNA gene fragment was then performed on these samples with primers P1/16S-SR followed by P1A/16S-SR (Deng, and Hiruki 1991; Lee et al. 2004), and two additional housekeeping genes (tuf and secA) were amplified as previously described (Makarova et al. 2012; Hodgetts et al. 2008; Bekele et al. 2011). All amplicons of the expected size, 1.5 kb (16S rRNA), 0.4 kb (tuf) and 0.6 kb (secA), were purified and bi-directionally sequenced using primers from each gene second round PCR amplification. Analysis of the sequences derived from the three gene fragments revealed no variation between the two plant samples and confirmed they originated from a phytoplasma, termed strain TXSB-2 (Texas Silver Bluestem). Sequences from a single B. laguroides plant DNA extract were deposited in GenBank with accession numbers OR711913 (16S rRNA), OR709687 (tuf) and OR709688 (secA). A BLAST search of the 16S rRNA gene sequence from TXSB-2 against the NCBI nucleotide database, showed 99.58% sequence identity with an unclassified phytoplasma clone 139-1 from a leafhopper collected in Australia (MW281491) (Fig. S2). The partial nucleotide sequence of the tuf and secA genes showed 90.60% and 89.78% similarity, respectively, to the corresponding genes in 'Ca. P. sacchari' strain SCWL1 (CP115156) associated with sugarcane in China. The iPhyClassifier, an interactive online tool for phytoplasma identification and classification (Zhao et al. 2009), was used to determine the 'Candidatus Phytoplasma' species affiliation and group/subgroup classification status of this phytoplasma strain. The result showed that the TXSB-2 16S rDNA shared 98.94% sequence identity with that of the 'Ca. P. sacchari' reference strain (GenBank accession: MN889545), indicating TXSB-2 is a 'Ca. P. sacchari'-related strain. The result from virtual restriction fragment length polymorphism (RFLP) analysis of the 16S rDNA F2nR2 fragment revealed that TXSB-2 possessed a collective RFLP pattern that is distinct from the reference patterns of all established phytoplasma ribosomal subgroups and is proposed as the representative strain of a new subgroup designated as 16SrXI-H. 'Candidatus Phytoplasma sacchari' has been reported associated with sugarcane grassy shoot disease, which is considered among the most damaging diseases of sugarcane across parts of Southeast Asia and India (Kirdat et al. 2021). The same phytoplasma was recently confirmed infecting sorghum in India (Nithya et al. 2024). To our knowledge, this is the first report of a 'Ca. P. sacchari'-related strain infecting B. laguroides in the United States. Moreover, B. laguroides is a new host for strains related to 'Ca. P. sacchari'. Further investigation is required to elucidate the prevalence of this disease in the area, its natural vectors, and the potential consequences arising from this novel phytoplasma strain within its ecosystem in Texas.

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.

Variability of phytoplasma associated with weeds grown in and around sugarcane crop in Uttar Pradesh, India

AbstractDuring survey from 2013 to 2018, Cynodon dactylon at Shahjahnapur, Azamagarh, Oplismenus burmannii at Shahjahnapur, Arundo donax at Deoria and Ocimum cannum at Gorakhpur were observed with phytoplasma suspected symptoms. The maximum disease incidence (34%) was recorded at Deoria District in case of A. donax plant with witches' broom symptom. The nested PCR using universal primer pairs (R16F2n/R16R2) confirmed the phytoplasma association with all the suspected samples. Further BLASTn and phylogenetic analysis of the sequences revealed the association of 16SrXIV-A subgroups phytoplasma with C. dactylon, O. burmannii and phytoplasma belonging to 16SrI-B subgroups with A. donax and O. cannum plants in the present study.

First report of 'Candidatus Phytoplasma asteris' associated with witches'-broom and plexus bud disease of Cerasus serrula in China.

Cherry blossoms (Cerasus serrula) are native to the temperate zone around the Himalayas in the northern hemisphere, mainly distributed in the west and southwest of China, including Yunnan, Sichuan and Tibet. Cherry has high ornamental, edible and medicinal value. In August 2022, we observed that Cherry trees exhibited witches' broom and plexus bud in Kunming City, Yunan Province, China. The symptoms consisted of many small branches with little leaves at the top of branches, stipule lobation, and clustered adventitious buds that are tumor-like on the branches that usually cannot sprout normally. As disease intensity increased, the branches dried up from the top to the bottom till the death of the whole plant. We named this disease C. serrula witches' broom disease (CsWB). We found CsWB in the areas of Panlong, Guandu, Xishan Districts in Kunming, where more than 17% of the plants we surveyed were infected. We collected 60 samples from across the three districts. These included 15 symptomatic and 5 asymptomatic plants per district. The lateral stem tissues were observed under a scanning electron microscope (Hitachi S-3000N). The nearly spherical bodies were found in the phloem cells of symptomatic plants. Total DNA extraction was conducted from 0.1 g tissue using the CTAB method (Porebski et al. 1997), ddH2O was used as the negative control, and Dodonaea viscose plants with witches' broom symptoms were used as the positive control. The nested PCR was used to amplify the 16S rRNA gene (Lee et al. 1993; Schneider et al. 1993) and PCR amplicon of 1.2 kb were amplified (GenBank accessions: OQ408098; OQ408099; OQ408100). The direct PCR specific to the ribosomal protein (rp) gene yielded amplicons of approximately 1.2 kb with primer pair rp(I)F1A and rp(I)R1A (Lee et a. 2003) (GenBank accessions: OQ410969; OQ410970; OQ410971). The fragment from 33 symptomatic samples was consistent with the positive control, and absent for asymptomatic samples, suggesting an association of phytoplasma with the disease. A BLAST analysis of the 16S rRNA sequences of CsWB phytoplasma showed that it has a 99.76% similarity with Trema laevigata witches' broom phytoplasma (GenBank accession MG755412). The rp sequence shared 99.75% identity with Cinnamomum camphora witches' broom phytoplasma (GenBank accession OP649594). An analysis with iPhyClassifier showed that the virtual RFLP pattern derived from the 16S rDNA sequence shares 99.3% similarity with that of the 'Ca. Phytoplasma asteris' reference strain (GenBank accession: M30790), and the virtual RFLP pattern derived from the fragment is identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (GenBank accession: AP006628). Thus, CsWB phytoplasma is identified as 'Ca. Phytoplasma asteris'-related strain belonging to sub-group 16SrI-B. The phylogenetic tree was constructed based on 16S rRNA gene and rp gene sequences by using MEGA version 6.0 (Tamura et al. 2013) with neighbor-joining (NJ) method and bootstrap support was estimated with 1000 replicates. The result indicated that the CsWB phytoplasma formed a subclade in 16SrI-B and rpI-B respectively. In addition, the clean 1-year-old C. serrula were tested positive for the phytoplasma using the nested PCR 30 days after being grafted with naturally infected twigs with CsWB symptoms. To the best of our knowledge, Cherry blossoms is a new host of 'Ca. Phytoplasma asteris'-related strains in China. The newly emerged disease is a threat to the ornamental value of cherry blossoms and the production of wood quality.

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

Pinus yunnanensis is an evergreen tree belonging to Pinaceae. The species is distributed in the east of Tibet, southwest of Sichuan, southwest of Yunnan, southwest of Guizhou and northwest of Guangxi. It is an indigenous and pioneer tree species for barren mountain afforestation in southwest China. P. yunnanensis has important value to both the building and medicine industries (Liu et al. 2022). In May 2022, P. yunnanensis showing witches'-broom symptom were found in Panzhihua City, Sichuan Province, China. The symptomatic plants had yellow or red needle, and exhibited plexus bud and needle wither. The lateral buds of infected pines developed into twigs. Some lateral buds grew in clusters and a few sprouted needles (Fig.1). The disease was named the P. yunnanensis witches'-broom disease (PYWB) and was found in some areas of Miyi, Renhe, and Dongqu. More than 9% of the pines showed these symptoms in the three areas surveyed, and the disease was spreading. A total of 39 samples were collected from three areas, including 25 symptomatic plants and 14 asymptomatic plants, respectively. The lateral stem tissues of 18 samples were observed under a scanning electron microscope (Hitachi S-3000N). Spherical bodies were found in the phloem sieve cells of symptomatic pines (Fig.1). Total DNA was extracted from 18 plant samples using the CTAB method (Porebski et al. 1997) and subjected to nested-PCR testing. Double-distilled water and DNA extracted from asymptomatic plants were used as negative controls, and DNA extracted from Dodonaea viscosa affected by the D. viscosa witches'-broom disease was used as positive control. Nested PCR was employed to amplify the pathogen's 16S rRNA gene (Lee et al. 1993; Schneider et al. 1993) and 1.2 kb segment were produced (GenBank accessions OP646619; OP646620; OP646621). PCR specific to the ribosomal protein (rp) gene yielded segment of approximately 1.2 kb (Lee et al. 2003)(GenBank accessions OP649589; OP649590; OP649591). The fragment size from 15 samples was consistent with the positive control, confirming the association of phytoplasma with the disease. A BLAST analysis of the 16S rRNA sequences of P. yunnanensis witches'-broom phytoplasma showed that it shared 99.12% ~99.76% identity with that of Trema laevigata witches'-broom phytoplasma (GenBank accession MG755412). The rp sequence shared 99.84%~99.92% identity with that of Cinnamomum camphora witches'-broom phytoplasma (GenBank accession OP649594). An analysis with iPhyClassifier (Zhao et. 2013) showed that the virtual RFLP pattern derived from OP646621 16S rDNA fragment of PYWB phytoplasma is identical (similarity coefficient 1.00) to the reference pattern of 16Sr group I, subgroup B (OY-M, GenBank accession AP006628). The phytoplasma is identified as a 'Candidatus Phytoplasma asteris'-related strain belonging to sub-group 16SrI-B. Interestingly, compared to AP006628, the virtual RFLP pattern derived from OP646619 and OP646620 fragments exhibit differences in three and one cleavage site, with a similarity coefficient of 0.92 and 0.97, respectively (Fig.2). These strains may represent a new subgroup within the 16Sr group I. The phylogenetic tree was reconstructed based on 16S rRNA and rp gene sequences using MEGA versio6.0 (Tamura et al. 2013). The analysis was conducted using the neighbor-joining (NJ) method with 1,000 replicates of bootstrap analysis. The results indicated that the PYWB phytoplasmas grouped into clades including phytoplasmas belonging to 16SrI-B and rpI-B, respectively (Fig.3). In addition, 2-year-old P. yunnanensis were used for grafting assays in nursery, and the twigs from infected pine under natural conditions were used as a scion, and the phytoplasma were detected using nested PCR after grafting for 40 d (Fig.4). In 2008-2014, P. sylvestris and P. mugo in Lituania had excessive branching symptoms that were attributed to 'Ca. Phtyoplasma Pini' (16SrXXI-A) or asteris' (16SrI-A) strains (Valiunas et al. 2015). In 2015, P. pungens with abnormal shoot branching in Maryland were found to be infected by 'Ca. Phytoplasma pini' strain (16SrXXI-B) (Costanzo et al. 2016). To the best of our knowledge, P. yunnanensis is a new host of 'Ca. Phytoplasma asteris'-related strain (16SrI-B) in China. The newly emerged disease is a threat to pines.

First report of a 'Candidatus Phytoplasma aurantifolia'-related strain associated with faba bean phyllody symptoms in India.

Vicia faba L. commonly known as broad bean or faba bean is one of the most widely grown protein rich legume crops. Out of more than 50 faba bean-producing countries, about 90% production is concentrated in the Asian, European Union (EU), and African region (FAO, 2020). Owing to its high nutritional value, both the fresh pods and dry seeds are consumed. During March 2022, some plants with little leaf and phyllody symptoms such as leaf-like floral structures were observed in the experimental fields of Indian Agricultural Research Institute (IARI), New Delhi (Fig. 1 a, b, c). The twig samples were collected from two individual symptomatic and one from asymptomatic plant. DNA was extracted using CTAB (cetyl trimethyl ammonium bromide) method (Ahrens and Seemüller, 1992; Marzachi et al. 1998) and examined for the association of phytoplasma through nested PCR using the universal primers P1/P7 and R16F2n/R16R2 targeting the 16SrRNA gene (Deng and Hiruki 1991; Gundersen and Lee 1996) and the other set of primers secAfor1/secArev3 and secAfor2/secArev3 targeting secA gene (Hodgetts et al. 2008). The DNA from symptomatic plants resulted the amplicons of 1200bp and 840bp specific to 16S rRNA and secA gene respectively. The gel purified PCR products were cloned into pGEM®-T Easy Vector system (Promega) and outsourced for Sanger sequencing at Agri Genome Labs, Kerala, India. The resultant 16S rRNA sequences (GenBank Acc. No. OP978231, OP978232) and secA sequences (ON715392 and ON715393) were examined through NCBI BLASTn analysis. The 16S rRNA sequences of the V. faba strains shared a minimum of 99.85% similarity with the phytoplasma strain causing little leaf and phyllody disease of sesame in India (MW622017) and a maximum of 100% sequence identity with the Vigna radiata phyllody and necrosis phytoplasma strain of Jodhpur (OP935760) India, whereas the secA gene sequences showed 100% identity with Tephrosia purpurea witches'-broom phytoplasma (MW603929) from China and a minimum of 91.14% similarity with 'Candidatus Phytoplasma aurantifolia' (MW020541) from India. The pairwise comparison results were completely in support of the corresponding phylogenetic sequence analysis results of 16SrRNA and secA gene sequences of faba bean strains in comparison with other strains retrieved from GenBank database, wherein the faba bean strains got clustered with 16SrII-D subgroup related strains (Fig. 2 a and b). Virtual RFLP analysis through iPhyClassifer tool through in silico digestion of R16F2n/R2 region of 16S rRNA gene of the faba bean strain using 17 restriction endonuclease enzymes resulted in the RFLP profiles similar to that of the profile of phytoplasma subgroup 16SrII-D (Y10097: papaya yellow crinkle) used as reference strain with a similarity coefficient value of 1.0. All the results of this investigation confirmed the association of 'Candidatus phytoplasma aurantifolia' (16SrII-D) with the diseased faba bean plants in this study. Previous reports of phytoplasma infecting faba bean include a group 16SrIII strain detected in Spain in 2004 (Castro and Romero), a subgroup 16SrII-D strain detected in Sudan in 2012 (Alfaro-Fernandez et al.), a group 16SrII strain detected in Saudi Arabia in 2014 (Al-Saleh and Amer), and subgroup 16SrIII-J strains detected in Egypt in 2014 (Hamed et al.) and in Peru in 2021 (Torres-Suarez et al.). To the best of our knowledge, these findings, document the first report of the association of 'Candidatus Phytoplasma aurantifolia' (subgroup 16SrII-D) with faba bean plants in India. This report necessitates further research on the status of distribution of this phytoplasma strain in other locations and hosts in the country so as to develop possible strategies to contain its further spread and management of the disease.

Phytoplasma association with phyllody of Sesamum species in India

Phytoplasma association with phyllody of Sesamum species in India

First Report of 'Candidatus Phytoplasma aurantifolia' Related Strain (16SrII-D) Associated with Stunting, Little Leaf and Phyllody Disease of Pearl Millet from South India.

Pearl millet [Pennisetum glaucum (L).R.Br.] also known as bajra, is one of the oldest millets and is cultivated in dry regions of arid and semi-arid tropics where no other cereal can be successfully grown. Pearl millet cultivation in India accounts for about two-thirds of millet production and is the fourth most cultivated food crop after rice, wheat and maize in India (Reddy et al. 2021a). In February 2021, the typical symptoms of stunting, phyllody and little leaf were observed after 25-30 days after sowing pearl millet seeds at Agricultural Research Station in Perumallapalle, Tirupati, India (Fig.1 A-C). The disease incidence was recorded up to 20% in the sampling regions. Total DNA was extracted from two symptomatic and two asymptomatic plant samples using CTAB DNA extraction method (Murray and Thompson, 1980). The extracted DNA was amplified in direct PCR and nested PCR assay using phytoplasma 16S rRNA universal primer pairs P1/P7 and R16F2n/R16R2 (Gundersen and Lee.1996) and secA gene with secAfor1/SecArev3 and SecAfor2/SecArev3 primer pairs (Hodgetts et al. 2008). 16SrRNA (1.25 kb) and secA (600 bp) gene amplicons were obtained from two symptomatic samples by nested PCR. No amplicons were produced with DNA from healthy leaf samples. Nested PCR amplified products (1.25 kb and 600 bp) from the symptomatic samples corresponding to the F2nR2 region of 16S rRNA and secA were directly sequenced at automated DNA sequencing facility (Eurofin Genomics India Pvt., Ltd Bangalore) and sequence data was deposited to NCBI GenBank with accession number ON005559 and ON067810. BLAST analysis revealed that pearl millet phytoplasma strain shared 100% sequence identity in 16Sr RNA and secA genes to 'Canditatus Phytoplasma aurantifolia' related strains (Acc. Nos. OM616883 and MT952965) from India. The subgroup was identified as 16SrII-D using the iPhyClassifier based on the virtual RFLP pattern derived from the query 16S rDNA F2nR2 fragment (Zhao et al. 2009). The virtual RFLP pattern is similar to the reference pattern of 16SrII-D (Y10096) with similarity coefficient 1.00. Phylogenetic analysis of 16S rRNA and secA gene sequences using MEGA version 7.0 revealed that the pearl millet phytoplasma strain clustered with 'Ca. P. aurantifolia' isolates of 16SrII-D subgroup. (Fig.1D-E) Earlier, one of 16SrI-B-phytoplasma strain (HM 134245) associated with green ear disease of pearl millet was reported in North India (Kumar et al. 2010). In this study, we reported the association of 16SrII-D subgroup phytoplasma with little leaves and witches'-broom disease of pearl millet in South India. Phytoplasmas belonging to the 16SrII-D subgroup have a wide range of hosts, including the agricultural and horticultural crops (Reddy et al., 2021b). Hence, this is the first report of 'Ca. P aurantifolia' infection in bajra in South India. The increase in the spread of 16SrII-D sub group phytoplasma diseases and the expansion of the host range strongly suggest further studies on the epidemiology of the dynamic dissemination of this disease in 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 asteris' (16SrI) from Cassia fistula showing symptoms of flat stem and witches'-broom in India.

Cassia fistula commonly known as 'golden shower tree' is a deciduous tree with a greenish-gray bark and complex leaves with lovely clusters of yellow blossoms that is also utilized for several purposes in traditional medicine offer therapeutic characteristics (Pawar et al., 2017). Random spotting of flat stem symptoms along with unopened flower beds was observed in C. fistula plant during March 2022 in IISER (Indian Institute of Science Education and Research), Thiruvananthapuram, Kerala, India and during May 2022 in SKUAST (Sher-e-Kashmir University of Agricultural Sciences and Technology), Jammu, which were suggestive of phytoplasma infection (Fig. 1 a-e). Surge of leaf hoppers was also observed in and around the tree. The leaf samples were collected from 3 individual C. fistula trees showing suspected symptoms of phytoplasma and one sample from asymptomatic plant of both the states. Leafhopper (LH) species were collected using sweep net method from both the locations. DNA was extracted using CTAB (Cetyl trimethyl ammonium bromide) method and nested universal PCR primers P1/P7 and R16F2n/R16R2 for the 16S rRNA gene (Deng and Hiruki 1991; Gundersen and Lee 1996) and secAfor1/secArev3 and SecAfor2/ SecArev3 for SecA gene (Hodgetts et al. 2008) were employed for the analysis of the phytoplasma strain association. The symptomatic plants and leaf hopper species showed positive bands of 1.2kb and 480bp for 16S rRNA and SecA gene respectively along with. Purified PCR products of both the genes (16Sr RNA and sec A) were ligated into pGEM ®T vector and cloned in Escherichia coli (DH5-α) were sequenced at Agri Genome labs, Kerala, India. The comparative sequence analysis using the BLASTn tool results showed 16S rRNA sequences acquired from plant samples (GenBank Acc. No. OP950857, OP950858) and the leafhoppers Hishimonus phycitis (OP538583) and Orosius albicinctus (OP538584) of Kerala had the minimum of 99.84% of similarity with Bitter gourd little leaf phytoplasma from Myanmar and maximum sequence identity (100%) with the Rapeseed phyllody phytoplasma strain from Taiwan. The sequences of phytoplasma strains from Jammu trees (Genbank Acc. No. OP801671 & OP801672) and H. phycitis (OP801673) shared 100% similarity with each other as well as with North American grapevine yellows and a minimum of 97.65% with Beta vulgaris phytoplasma from Poland. The pairwise comparison results were completely supported by the corresponding phylogenetic sequence analysis of 16S rRNA and SecA gene sequences of all the isolates in the study which clustered with 16SrI-B subgroup related strains. Virtual RFLP analysis through iPhyClassifer results that were derived from in silico digestions of R16F2n/R2 region of 16S rRNA gene using 17 restriction endonucleases enzymes indicated that all the samples produced similar virtual RFLP profiles identical to the reference strain of 16SrI-B phytoplasma subgroup (aster yellows: Acc. No. M30790) with a similarity coefficient value of 1.0. To the best of our knowledge, this is the first report of the phytoplasma association of 'Ca. P. asteris' (16SrI-B) subgroup with Cassia fistula in the world.

Lethal yellowing disease in <em>Borassus flabellifer</em> in Jaffna peninsula

Palmyrah palm (<em>Borassus flabellifer </em>Linn.) is a highly versatile tree mainly distributed in the North and Eastern provinces of Sri Lanka. Even though the plant is rarely affected by pathogens due to its extremely woody nature, some significant abnormalities in plants growing in the Pungudutivu area in Jaffna Peninsula have been noted recently. The present study aimed to characterize the abnormality, identify the disease and confirm the possible causative agent. In this study, the progression and incidence of disease were determined through field visits. A literature search was conducted to compare the symptoms with other palm varieties. Additionally, the possibilities for phytoplasma infection were tested with phytoplasma-specific universal primers using PCR. Initially, the plants showed progressive leaf yellowing and drying from the leaf tip towards the petiole followed by collapsed spear leaf hanging downward in the crown. Finally, the crown falls off the trunk. The symptoms are characteristics of the lethal yellowing disease. The PCR result confirmed the association of phytoplasma with the diseased plants. To our knowledge, this is the first report from Sri Lanka regarding the lethal yellowing disease in Palmyrah palm. Since the disease severely affects the plant and spreads rapidly, there is an urgent need to study the epidemiology of the disease and to find out suitable control measures.

Evidence of mixed infection of phytoplasma and begomovirus associated with Withania somnifera and Capsicum annum plants from Uttar Pradesh, India

Symptoms of yellow discoloration, curling of leaves, and little leaves with excessive branching were observed on Withania somnifera and Capsicum annum plants, with an average incidence of 5 and 8%, respectively, in Hardoi District of Uttar Pradesh, India during 2016–17 and 2017–18. The little leaf and excessive branching suggested the possibility of phytoplasma association. However, the presence of whiteflies in the vicinity and leaf curling suggested begomovirus infection. Three leaf samples from Withania ­somnifera and Capsicum annum symptomatic plants and one from non-symptomatic leaf were used for DNA isolation and were subjected to PCR using P1/P6 primers and nested PCR R16F2n/R16r2 primers, respectively, for the detection of phytoplasma. The begomovirus coat protein-specific primer AV1F/AV1R was used to detect begomovirus infection. Nested PCR amplified the ∼1.2 kb amplicon in all six symptomatic leaves and no amplification was observed in non-symptomatic leaves. The CP region primer yielded ∼800 bp amplicons in all three symptomatic samples of each plant. Amplified products from both primers were eluted, purified, and sequenced. The phytoplasma sequence obtained from Withania somnifera (MH789552) shared the highest sequence identity (99.92%) with other isolates of a clover proliferation group (16SrVI-D) group of phytoplasmas. However, Capsicum annum plants shared the highest identity (99%) with the Ca. P. asteris-related (16SrI-B) group of phytoplasma. In silico RFLP analysis of the 1.2 kbp product of the 16S rRNA sequence of the W. somnifera and C. annum phytoplasma strains submitted to the pDRAW32 tool (https://www.acaclone.com/) and phylogenetic analysis through the MEGA 6.0 tool confirmed that it as a member of the 16SrVI-D subgroup and 16SrI-B subgroup, respectively. The CP gene sequence of the W. somnifera isolate (MW176071) showed maximum identity (99%) with several isolates of the Tomato leaf curl virus reported from various places in India. However, the C. annum isolate (MW420480) shared maximum identity with the Ageratum enation virus. The investigation confirmed the mixed infection of ToLCV and 16SrVI-D group phytoplasma in the W. somnifera plant and 16SrI-B alongwith AEV in C. annum plants.

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 Australasia Associated with Alfalfa Witches’ Broom: Symptomatology, Quantitative Loss, Qualitative Loss and Molecular Characterization

Background: Alfalfa, Medicago sativa L. is the most important and widely grown leguminous fodder crop in temperate and tropical regions of the world. The production of alfalfa crop is limited by several biotic stresses, among which witches’ broom disease (AWB) was reported to cause significant economic losses. Methods: The phytoplasma infected alfalfa plants were collected from a central research farm, ICAR-IGFRI, Jhansi, U.P. Qualitative parameters such as crude protein content, acid detergent fibre and neutral detergent fibre were estimated in diseased and healthy plants. Phytoplasma universal primer (P1/P7) and nested primer (R16mF2/R16mR1) were used for the molecular characterization of AWB infected plants and phytoplasma infected, Parthenium hysterophorus. Result: The incidence of AWB disease ranged from 8-10%. The quantitative analysis of disease plants showed reduced plant height (-35%), fresh weight (-46.89%) and dry weight (-50.08%) compared to healthy plants. The diseased plant recorded low crude protein content (-21.38%) and higher dry matter content (+0.68%), acid detergent fibre (+33.72%) and neutral detergent fibre (+13.06%). The association of phytoplasma in diseased alfalfa and parthenium samples was confirmed by using P1/P7 and R16mF2/R16mR1 primer pair and Blastn analysis shared 99.6-100% similarity with ‘Candidatus Phytoplasma australasia’ belongs to the 16Sr group II-D.

Detection and characterization of diverse phytoplasmas in ornamental and cultivated pomegranate species in Turkey

Yellowing, leaf curling and shoot-dieback symptoms on pomegranates have been observed in Turkey in last decade, however, the association of phytoplasmas to symptoms in pomegranates has been not fully understood. In the present study, 130 cultivated and 65 ornamental pomegranate trees were sampled from three provinces (Hatay, Adana, Sanliurfa) from southern part of Turkey. Nested-PCR assay and restriction fragment length polymorphism analysis were employed to identify the phytoplasma strains detected in symptomatic and asymptomatic pomegranate trees. Among 195 tested samples, three ornamental and six cultivated pomegranates were found positive for phytoplasmas by Nested-PCR. Based on virtual RFLP analysis of 16S rDNA sequences, the strains detected in cultivated pomegranates were related to ‘Candidatus Phytoplasma asteris' (16SrI–S), ‘Ca. P. solani’ (16SrXII-A) and ‘Ca. P. mali’ (16SrX-A) whereas in the ornamental pomegranates only to ‘Ca. P. prunorum’ (16SrX-B) was detected. The association of ‘Ca. P. mali’ and ‘Ca. P. asteris’ related strains on cultivated pomegranates and as well as ‘Ca. P. prunorum’ in ornamental pomegranates have been identified for the first time worldwide. The presence of distinct phytoplasmas in pomegranates could result a high diversity of phytoplasmas within single plant species.

Detecção de 'CA. Phytoplasma phoenicium' em plantas de pervinca (Cathranthus roseus) em Uttar Pradesh, Índia

Cathranthus roseus also known as periwinkle, an ornamental plant contains several medicinal values, was found with the symptoms of little leaf and witches’ broom at Shahjahanpur location with the incidence of up to 8%. The phytoplasma etiology was confirmed through scanning electron microscopy examination in all the four-leaf samples. Molecular analysis through PCR with universal primer pairs P1/P6 followed by nested PCR with R16F2n/R16r2 primers yielded ~1.2kbp amplicons in all the four symptomatic leaf samples. One amplicon was eluted, purified, sequenced, and used in BLASTn searches, which showed maximum identity of periwinkle isolate with several isolates of 16SrIX group of phytoplasma. Further, phylogenic analysis and in silico RFLP confirmed the association of 16SrIX-C subgroup phytoplasma in little leaf and witches broom plants which is the first report from India.

First Report of 'Candidatus Phytoplasma trifolii' Associated with Little Leaf and Floral Virescence Disease in Solanum violaceum from the North East Region of India.

Solanum violaceum Ortega, (Indian nightshade), is a medicinal plant belonging to the family Solanaceae. It grows naturally as a weed in agricultural fields, forest edges, road sides and fallow lands throughout the North East Region of India. It is mostly consumed as a vegetable by the native people of the region because of its promising therapeutic effects (Islam and Islam 2018). In November 2019, typical phytoplasma-suspected symptoms such as little leaf, yellowing and floral virescence were observed on S. violaceum plants (Figure 1) in Kaliabor, Nagaon district of Assam, India (26.3220'N, 92.5540'E), with about 8% incidence based on visual observations. To investigate the possibility of a phytoplasma association with the symptoms, total DNA was isolated from collected leaf samples (symptomatic-6 and asymptomatic-9) by following the CTAB protocol (Kollar et al. 1990). The DNAs isolated were assayed for the presence of phytoplasma using polymerase chain reaction (PCR) assays performed with P1/P6 primer pair for 16S rRNA gene (Deng and Hiruki 1991) and SecAfor1/SecArev3 for secA gene (Hodgetts et al. 2008). The direct PCR targeting the 16S rRNA gene and secA gene amplified a product of about 1.5 kb and 840 bp, respectively, from all the symptomatic plant samples but not from any of the asymptomatic plant samples. All amplicons were double strand sequenced and corresponding high quality sequences were deposited in the GenBank with accession numbers MW261863 for 16S rRNA gene and MW885174 for secA gene with a sequence length of 1406 bp and 532 bp, respectively. Pairwise sequence comparison of 16S rRNA gene sequence of S. violaceum phytoplasma isolate revealed 100% sequence identity with strain of 'Candidatus Phytoplasma trifolii' (Accession number, EF186820) and secA gene showed up to 95% sequence identity with the same organism (Accession number, KX784498). Further analyses of the 16S rRNA and secA genes based phylogenetic tree (Figure 2A and B) and the iPhyClassifier-based virtual RFLP analysis of 16S rRNA gene revealed that the phytoplasma-associated with little leaf and floral virescence of S. violaceum belongs to 16SrIV-D subgroup with a similarity coefficient of 1.0. The 16S rRNA and secA gene sequences comparison confirmed the close association of phytoplasma strain associated with S. violaceum with 16SrVI-D subgroup phytoplasma. Earlier, the 16SrVI-D subgroup of phytoplasma has been reported to be associated with many horticultural and other agricultural crops in India (Rao 2021). Recently, the 16SrVI-D strain was reported in eggplant from Assam, India (Accession number, MW261866), showing up to 100% sequence identity with S. violaceum strain (Accession number, MW261863) (Dutta et al. 2020). To the best of our knowledge, this is the first report of the association of the 16SrVI-D subgroup of phytoplasma strain with S. violaceum plant, which is an important medicinal plant widely used in folk and traditional Indian systems of medicine. Since this study has confirmed S. violaceum as a new host of 16SrVI-D group of phytoplasma, further studies on the disease epidemiology and insects vectoring the phytoplasma would help formulate effective management strategies in preventing further spread to other hosts.

First report of 16SrII group (Peanut witches' Broom) Phytoplasmas associated with the Leucas aspera Phyllody in India.

Leucas aspera (Wild.) Linn. (Family: Lamiaceae) is a commonly found weed throughout India, known for its pharmacological properties. Its white flowers and leaves are used in many Ayurvedic formulations for the treatment of chronic rheumatism, psoriasis, snake bites and skin eruptions (Prajapathi et al., 2010). During a survey of commercial flower crop fields in May 2018, a few L. aspera plants, growing as unwanted weeds in the fields and surrounding agricultural wastelands with the symptoms of phyllody, virescence and little leaves were observed in Emmekoppalu (12.2106, 76.2511; n= 1/26 plants) and Beerihundi (12.1630, 76.3225; n= 2/59 plants) localities of Mysuru district, and Srirangapatna in Mandya district (12.2541, 76.411; 1/67 plants), Karnataka- India(Figure 1). 'n' denotes the symptomatic/ asymptomatic samples observed. The disease incidence in the surveyed localities ranged less than four per cent. The total genomic DNA was extracted from the leaf midrib tissues of three representative symptomatic and two asymptomatic samples using the CTAB method. The phytoplasma 16S rRNA gene was amplified in nested PCR assay by P1/P7 followed by R16F2n/R16R2 primers using Long Amplification (LA) Taq polymerase (Takara, Japan). Additionally, the PCR assays were performed for the amplification of phytoplasma secA gene using the primers SecAfor1/SecArev3 and SecAfor2/SecArev3 (Hodgetts et al., 2008). The DNA templates from all the symptomatic samples generated amplicons of approximately 1.25kb (16S rRNA gene) and 480 bp (secA gene) revealing the association of phytoplasma strains. No amplifications were observed for the asymptomatic L. aspera samples. The obtained 16S rRNA gene sequences (MN223676, MT807111 and MZ093053) showed 97.96, 98.37 and 98.18 % sequence identity, respectively; with the 'Candidatus Phytoplasma aurantifolia', strain 'WBDL (U15442) using EzBiocloud database. The NCBI-BLAST analysis revealed maximum identity to various Peanut witches' Broom (PWB) phytoplasma strains. The virtual RFLP tool, iPhyClassifier delineated the Leucas phyllody phytoplasma strains (MN223676, MT807111 and MZ093053) to group 16SrII (PWB, Peanut Witches' broom group) subgroup D with the similarity coefficient 1.0 (Zhao et al. 2009). The obtained secA gene sequences (MZ151944, MZ151945 and MZ151946) were 98.15 to 100 % similar to the strain sequences of PWB phytoplasma strains. Further, the clustering pattern in the phylogenetic trees (16S rRNA and secA genes) constructed using MEGA 7 confirmed that the Leucas phyllody phytoplasma sequences were closely related to PWB strains. To the best of our knowledge, this is the first report on the association of 16SrII-D subgroup phytoplasma with the phyllody disease of L. aspera. In India, many weeds and wild plants serve as alternative hosts of PWB phytoplasmas and aid in the emergence of related diseases in economically important crops (Thorat et al., 2016; Thorat et al., 2017). The close genetic association of phytoplasma strains found in L. aspera and many other crops indicates the presence of common insect vector(s) transmitting these phytoplasmas (Yadav et al. 2015). This report is an addition to the catalogue of the weed species harboring phytoplasma strains associated with economically important crop plants (Rao et al., 2017). The screening of phytoplasma strains in weeds, alternate hosts and known/ unknown insect vectors is therefore essential to develop management strategies and effective management of phytophagous insect vectors feeding on both weeds and crop plants.