Virtual Restriction Fragment Length Polymorphism Research Articles

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 asteris'-related Strain (16SrI group) Associated with Small Leaves, Leaf Yellowing, and Shoot Proliferation of Morning Glory (Ipomoea biflora) in Taiwan.

Ipomoea biflora L., commonly known as morning glory, is an herbaceous vine plant in the Convolvulaceae family and is widespread at low elevations in Taiwan and other East Asian countries. In September 2023, six I. biflora plants exhibiting small leaves, leaf yellowing, and shoot proliferation were observed in a vacant lot in Taiwan Agricultural Research Institute (TARI), Wufeng District, Taichung, Taiwan, representing 100% disease incidence in the area. All the symptomatic morning glory climbed onto Murraya paniculata L. (common jasmine orange) which however showed no similar symptoms. The total DNA (two samples for each plant) from leaf tissues of three symptomatic morning glory plants, two asymptomatic morning glory plants, and one asymptomatic common jasmine orange was isolated by the CTAB method (Fulton et al. 1995) and used for PCR with the universal primers, P1 (Deng and Hiruki 1991)/P7 (Schneider et al. 1995), to amplify a fragment containing partial 16S rDNA. Expected 1.8-kb bands were amplified from DNA extracted from all symptomatic plants, whereas no PCR product was detected from that of the asymptomatic I.biflora and M. paniculata plants. Six PCR products were cloned and sequenced in the Biotechnology Center DNA-sequencing facility at National Chung Hsing University, and one representative sequence was selected and deposited in GenBank. BLAST analysis revealed that the obtained 16S rDNA sequence (PP230905) shared 99.92% identity with the following phytoplasma strains: rapeseed phyllody phytoplasma (CP055264), plumbago auriculata leaf yellowing phytoplasma (MN239503), and aster yellows phytoplasma (MK992774), which all belong to the 16SrI subgroup. The query 16S rDNA sequence shares 99.84% identity with that of the 'Candidatus Phytoplasma asteris' reference strain (M30790), suggesting that the phytoplasma is a 'Ca. Phytoplasma asteris'-related strain. A virtual restriction fragment length polymorphism (RFLP) analysis was conducted using iPhyClassifier tool (Zhao et al. 2009), and the pattern derived from the 16S rDNA fragment of the I. biflora phytoplasma was identical (similarity coefficient 1.00) to the reference pattern of 16SrI, subgroup B (onion yellows phytoplasma OY-M; AP006628). Six total DNA samples from symptomatic plants were used as templates to amplify 842 bp secA sequences with SecAfor1 and SecArev3 primers (Hodgetts et al. 2008), and one representative sequence was deposited in GenBank. The partial secA sequence (PP263636) showed 98.22% identity with that of Trema levigatum witches'-broom phytoplasma (MW032212) that also belongs to the 16SrI group (Wan et al. 2021). Phylogenetic analysis of both 16S rDNA and secA confirmed I. biflora phytoplasma as 16SrI, subgroup B. Taken together, we concluded that the morning glory phytoplasma in this study was a 'Ca. Phytoplasma asteris'-related strain belonging to the 16SrI group. To the best of our knowledge, this is the first report of a phytoplasma-infected I. biflora in Taiwan, suggesting morning glory as a new natural host of 16SrI phytoplasmas, alongside other plants like roselle and citrus (Tseng et al. 2014; Feng et al. 2015).

Ampelopsis grossedentata Represents a New Host of the 16SrI Group of Phytoplasma Associated with Yellow Leaf Symptoms in China.

Ampelopsis grossedentata, commonly known as "Vine Tea" and well-recognized for its rich flavonoid content, is mainly distributed in the southern regions of the Yangtze River basin in China. These regions include Hunan, Hubei, Jiangxi, and Guizhou provinces. Vine Tea is mainly consumed as an herbal tea and has garnered attention for its reported health benefits, including antioxidant, anti-inflammatory, anti-tumor, anti-diabetic, and neuroprotective properties. It has been used to alleviate coughs and sore throats (Zhang et al., 2021; Wang et al., 2017; Gao et al., 2009). In the Zhangjiajie region of Hunan province alone, the Vine Tea planting area reached 7,670.5 hectares and produced commercial goods worth 1.417 billion RMB in 2022. In May 2021, leaf margins and veins fading to yellowing mottling, and crumpling of leaf blades in the shape of a boat symptoms were found in ~16% of Vine Tea plants in the Sanjiakuan Township, Yongding District, Zhangjiajie region (29°15'E, 110°30' N) (Figure 1a, b, c). (Figure 1a, b, c). Phytoplasma-like microbial cells (small oval shaped bacterial cells, around 1000 nm in size) were observed in sieve tube cells in the phloem of diseased leaves using transmission electron microscopy. No such cell was observed in the phloem of healthy leaves (Figure 2a, b). To investigate the potential association between phytoplasma and the observed symptoms of the diseased plants, total DNA was isolated from ten diseasedeaves and compared with ten healthy leaves from the same field using SteadyPure Plant Genomic DNA Extraction Kit. The isolated DNAs were analyzed first in a direct PCR using universal phytoplasma primer pair R16mF2/R16mR1 targeting the 16S rRNA gene (Gundersen and Lee 1996) and specific pair rpF1/rpR1 (Lee et al. 1998) targeting the DNA fragment encoding partial ribosomal proteins (rp) L22 (complete) and S3 and S19 (partial). The initial amplified products were used as templates and further amplified by nested PCR respectively with primer pair R16F2n/R16R2 for the 16S rRNA gene (Lee et al. 1998) and the rpF2/rpR2 primer pair for the rp gene (Martini et al. 2007). No amplification was obtained with DNA from healthy leaf samples using any of the four primer pairs. The amplified fragments from diseased leaves by nested PCR were cloned and sequenced (Qingke Biotech, China). The obtained sequences have been deposited in GenBank with accession numbers OR282806 for the 16S rRNA gene and GenBank OR353012 for the rp gene. BLASTn analysis revealed that the partial 16S rRNA gene sequence in our sample shared 99.4% nucleotide sequence identity with 'Candidatus Phytoplasma sp.' (MW364378) and 'Peony yellows phytoplasma' (KY814723) of the 16SrI group. Similarly, our rp gene sequence shared 99.6% nucleotide identity with the rpI group of phytoplasma such as the 'Balsamine virescence phytoplasma' (JN572890) and 'Paulownia witches'-broom phytoplasma' (HM146079). Phylogenetic analysis of the 16S rRNA and rp sequences using MEGA version 7.0 revealed that the phytoplasma strain associated with A. grossedentata yellow leaf syndrome in our study site belonged to the 16SrI (Candidatus Phytoplasma asteris) group of phytoplasma (Figure 3a, b). Using the interactive online phytoplasma classification tool iPhyClassifier (Zhao et al., 2009), virtual restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene sequences showed our strain having a distinct RFLP map but was closest to that of the onion yellow phytoplasma 16SrI-B subgroup (GenBank accession number: AP006628), with a similarity coefficient of 0.94 (Figure 4a, b). To confirm phytoplasma transmission, healthy plants were inoculated with three scions of infected plants of A. grossedentata. After 16 days, the new leaves of the inoculated A. grossedentata showed yellow leaf symptoms (Figure 5a, b, c), akin to the symptoms originally observed in the field, and the outer contour of the leaf margin appeared chlorotic. After 26 days, primer pairs R16mF2/R16R1 and R16F2n/R16R2 were used for nested PCR detection of phytoplasma in symptomatic A. grossedentata leaves. Phytoplasma was detected in the first and second leaves of symptomatic branches and leaves while negative control showed no amplification. Sequencing of the amplified fragments showed 100% nucleotide identity to the strain from the grafting source. Our results indicated that the pathogen and the disease can be transmitted by tissue grafting, consistent with the biological characteristics of phytoplasma, and further confirmed that the phytoplasma was the pathogen of yellow leaf syndrome of A. grossedentata. Toour knowledge, this is the first report of phytoplasma of group 16SrI affecting A. grossedentata.

First Report of 'Candidatus Phytoplasma solani' (16SrXII-A) Associated with Fox Nut (Euryale ferox Salisb.) Stolbur Disease in India.

Fox nut also known as Gorgon nut, Makhana (Euryale ferox Salisb.) is a high value aquatic crop belonging to the family Nymphaeaceae. In India, it is generally grown in flood prone areas of North Bihar, lower Assam, parts of West Bengal, Odisha, Manipur and Tripura (Jana et al., 2018). India contributes nearly 70-80% of the global fox nut production. During September 2021, a phytoplasma like symptom was noticed on fox nut leaves at Basudeopur Farm of Research Centre for Makhana, Darbhanga, Bihar, India (23° 9' N and 65° 53' E). The characteristic symptom was that some portion of leaf lamina deformed along the veins with wrinkled and raised overgrowth or hypertrophy. The veins were thickened and reddened in the infected leaf area. The infection occurs in petiole as well as in flower stalk. The disease incidence was found as high as 30% which caused severe yield loss which was calculated to be 40% in that particular field. Total of 20 sampled fox nut plants, 10 symptomatic and 10 asymptomatic ones, were collected and tested for the presence of phytoplasma. A nested PCR assay using the phytoplasma universal 16S rRNA primer pairs: P1/P7 followed by R16F2n/R16R2 (J. Jović et al. 2011) amplified the expected ~1.2-kb 16S rDNA fragment in all 10 symptomatic samples. No amplification was detected from asymptomatic samples. One of the ten 1.2-kb nested 16S rDNA PCR products was gel purified, cloned into the pGEM-T-easy plasmid vector (Promega, Madison, WI), and sequenced and was deposited in NCBI under the Accession no.OL873590. BLAST analysis showed that the sequence of the PCR 16S rDNA product was 100% identical to several GenBank sequences of Ca. P. solani (16SrXII Stolbur group) viz. KF907506. Furthermore, analysis by iPhyClassifier software showed that the virtual restriction fragment length polymorphism (RFLP) pattern of the sequenced PCR 16S rDNA product is identical (similarity coefficient 1.00) to the reference pattern of the 16SrXII-A subgroup. Identification of 'Ca. P. solani' was conducted following the STOL11 stolbur-specific protocol (Radonjić et al. 2009). Sequencing of tuf gene (Elongation factor Tu) was performed by using tuf marker genes (Cvrković et al. 2014) from 10 symptomatic and 10 asymptomatic samples. Sequence of the amplified gene (896 bp) was deposited in GenBank under Accession number OM174272. The presence of 'Ca. P. solani' was detected in all symptomatic samples, while all control plants tested negative. The RFLP analysis of tuf gene nested PCR products using HpaII endonuclease (Fermentas) revealed uniform tuf-b type in all positive samples. Nucleotide blast analyses showed that the tuf gene was 100% identical to STOL11 strain of C. P. solani subgroup 16SrXII-A (Accession No JQ797670). For developing a suitable management strategy, identification of the vector is essential. Leaf hoppers visiting the infected plants as well as nearby crop fields will be tested for presence of the phytoplasma. To the best of our knowledge, this is the first report of Candidatus Phytoplasma solani' (16SrXII-A) infecting Fox nut (Euryale ferox Salisb.) in India. References Cvrković et al. 2014. Plant Pathol. 63:42. https://doi.org/10.1111/ppa.12080 Jana, B. R., et al. 2018. Int. J. Curr. Microbiol. App. Sci. 7(12): 578-587. https://doi.org/10.20546/ijcmas.2018.712.072 Jović, J. et al. 2011. B. Insectol. 64:S83. ISI Radonjić, S. et al. 2009. J. Phytopathol. 157:682. https://doi.org/10.1111/j.1439- 0434.2009.01560.

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.

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 ‘Candidatus Phytoplasma australasia’-related strain’ (16SrII-D) in Pyracantha angustifolia (narrowleaf firethorn)

Little leaf and yellowing symptoms associated with phytoplasma disease were observed in Pyracantha angustifolia in Turkey. Amplification (through nested PCR) and virtual restriction fragment length polymorphism (V-RFLP) confirmed the presence of phytoplasma in the symptomatic plants the phylogeny based on the R16F2n/R2 sequences of the P. angustifolia phytoplasma was established and the sequence analysis showed that the isolate had 98.7% similarity to ‘Candidatus Phytoplasma australasia’, a member of the 16SrII-D subgroup. This is the first world report of a phytoplasma in P. angustifolia and its first identification in the Republic of Türkiye.

Application of 16S rRNA virtual RFLP for the discrimination of some closely taxonomic-related lactobacilli species

BackgroundSeveral species in Lactobacillaceae family were recognized as potential probiotic bacteria. In this group of lactic acid bacteria, species are taxonomically closed and usually share similar 16S rRNA gene, thus, instead of so their identification and discrimination are too difficult. MethodIn the present study, virtual restriction fragment length polymorphism (RFLP) is instead of was used as a tool to discriminate between the closely related species Lactiplantibacillus plantarum (L plantarum), Lactiplantibacillus paraplantarum (L paraplantarum), and Lactiplantibacillus pentosus (L pentosus); Latilactobacillus sakei (L sakei), Latilactobacillus curvatus(L curvatus), and Latilactobacillus graminis (L graminis); Lacticaseibacillus casei (L casei), Lacticaseibacillus paracasei (L paracasei), Lacticaseibacillus zeae, and Lacticaseibacillus rhamnosus; Lactobacillus gasseri (L gasseri) and Lactobacillus johnsonii (L johnsonii). In silico comparative analysis of 16S rRNA sequences digested by 280 restriction enzymes was performed in order to search the key enzymes which gives different profiles. ResultsResults revealed that L casei, L paracasei, L zeae, and Lb rhamnosus could be separated from each other on the basis of AlwI, BpuEI, BsgI, BsrDI, BstYI, EarI, MluCI, and NsPI RFLP. Results showed also that different RFLP patterns were obtained from L sakei, L graminis and L curvatus by using both AflI and NspI endonucleases (in separated restriction) and L plantarum, L paraplantarum, and L pentosus were distinguished each one from the other by MucI, NspI, and TspDTI PCR-RFLP. Lb gasseri and L johnsonii were also separated on the basis of Mse I, Taq I, and Dra I RFLP. ConclusionIn this study, we proved that too closely related species could be separated in virtual analysis on basis of their 16S rRNA RFLP patterns using key restriction enzymes method.

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.

Detection, Identification, and Molecular Characterization of a 16SrII-V Subgroup Phytoplasma Associated with Nicotiana plumbaginifolia.

Nicotiana plumbaginifolia Viviani, commonly known as curl-leaved tobacco, is an annual herbaceous plant belonging to Solanaceae family. This plant is native to Mexico, South America, and parts of the Caribbean and has been reported to be present in Taiwan since 2006. In March 2021, N. plumbaginifolia Viviani, found in Yunlin County, Taiwan, was observed to have phyllody, virescence, and witches'-broom, which is consistent with the disease symptoms caused by phytoplasma infection. Samples of the healthy and symptomatic plants were collected for analysis of the causal agent associated with the diseased N. plumbaginifolia Viviani. Under transmission electron microscopy, the phytoplasma-like pleomorphic bodies were found in the sieve tubes of the diseased plants. The 16S ribosomal RNA (rRNA)-based phylogenetic analysis and the iPhyClassifier-based virtual restriction fragment length polymorphism study demonstrated that the phytoplasma identified in this study can be classified into the 16SrII-V subgroup, which is similar to the peanut witches'-broom phytoplasma, a 'Candidatus phytoplasma aurantifolia'-related strain. Further identification of SAP54/PHYL1 and SAP11 homologs in the phytoplasma explain the disease symptoms of phyllody, virescence, and witches'-broom observed in diseased N. plumbaginifolia Viviani. The discovery of new phytoplasma plant hosts has gained scientific importance in light of the attempt to unravel an efficient strategy to fight the rapid spread of this disease, which poses a threat to the agricultural sector and food security in Taiwan.

New genetically distinct phytoplasmas and insect carriers associated with pine tree disease revealed by a survey in the Curonian Spit, Lithuania

Our previous studies reported that phytoplasma was the causative agent of the pine disease in the Curonian Spit, Lithuania. In this study, insects from diseased pine trees and their adjacent areas were collected from 2016 to 2019 to further identify potential insect vectors that spread phytoplasmas. A total of 1018 phloem-feeding insects (order Hemiptera) were identified, 98.62% of which were aphids (Aphididae), and no known phytoplasma vectors were found. The results from semi-nested polymerase chain reaction (PCR) using phytoplasma-specific universal primers revealed that phytoplasmas were detected in Scots pine aphids (Cinara pini (Linnaeus, 1758)), waxy grey pine needle aphids (Cinara pineti (Fabricius, 1781)), and species-unknown aphids. Further sequence analysis and virtual RFLP (restriction fragment length polymorphism) analysis of aphid-harbored phytoplasma strains indicated that they were closely related to ‘Candidatus Phytoplasma pini’ (16SrXXI-A), but mainly 16SrXXI-A variants, which were also main strains identified in diseased pine trees. In addition, three new phytoplasma subgroups were delineated in the present study. Subgroups 16SrXXI-C and 16SrXXI-D were unveiled from previously identified (but classification was overlooked) Lithuanian pine phytoplasma strains. Subgroup 16SrXXI-E was discovered from the newly identified aphid-harbored phytoplasmas. Further transmission trial studies on these aphids will provide insights into the epidemiology and pathosystem of pine phytoplasma diseases, as well as disease management.

Candidatus phytoplasma malaysianum (16SrXXXII) associated with Elaeocarpus sylvestris decline in South Korea

Phytoplasma has been newly detected in Elaeocarpus sylvestris, a tree species that grows on the island of Jeju, South Korea. Various symptoms of infection of E. sylvestris have appeared since 2013, including loss of leaf coloration, such as yellowing, darkening and branch dieback. We analyzed leaves and seeds of E. sylvestris sequences infected by phytoplasma, and found partial 16S rRNA sequences to be 99.5%, and secA genes 98.2% correlated with Malaysian periwinkle virescence group strain ELY-BN1, MaPV, MOP, MYD, and TtWB. Analyzing these sequences and through the virtual restriction fragment length polymorphism (virtual-RFLP), we report here the first occurrence of Elaeocarpus decline associated with a phytoplasma strain belonging to 16SXXXII groups, as well as the first detection of phytoplasma in the seeds of E. sylvestris in South Korea.

Detection, in silico analysis and molecular diversity of phytoplasmas from solanaceous crops in Turkey

Phytoplasma-like symptoms of leaf yellowing and calyx malformation were observed in eggplant (Solanum melongena L.), upward leaves and fruit malformation in pepper (Capsicum annuum L.), and aerial tuber formation in potato (S. tuberosum L.) during the survey performed in the late season (August to September) of 2015 and 2016 in Van province (Turkey). A total of 100 samples were tested by nested-PCR using universal primer pairs to assess the sanitary status of the solanaceous crops and to characterise the phytoplasma isolates. Among them, seven samples resulted in a 1.25 kb DNA fragment, and five (two eggplants, two peppers, and one potato) were molecularly characterised (Accession No.: KY579357, KT595210, MF564267, MF564266, and MH683601). BLAST and the virtual restriction fragment length polymorphism (RFLP) analysis of 16S rRNA genes revealed the presence of two distinct phytoplasma infections in solanaceous crops: ‘Candidatus Phytoplasma trifolii’ a member of the clover proliferation group (16SrVI) and subgroup A and ‘Candidatus P. solani’ a member of the stolbur group (16SrXII) and subgroup A. The virtual RFLP analysis and calculated coefficients of RFLP pattern similarities further revealed a remarkable genetic diversity among the ‘Candidatus P. solani’ isolates infecting pepper (similarity coefficient of 0.90) and eggplant (similarity coefficients of 0.98 and 1.00) at the same geographical area. This is the first report of the natural occurrence of ‘Candidadtus P. trifolii’ in potato from the Eastern Anatolia region, Turkey.

Identification of 16SrXXX-A Phytoplasma Associated with Salt cedar (Tamarix chinensis Lour.) witches'-broom (SCWB) in Southern Xinjiang of China.

Salt cedar is an ornamental shrub/moderate tree species native to Asia and East Europe, and grows in salt-alkali soil, desert and other dry areas, which plays an important role in wind prevention and sand fixation as well as maintaining ecological balance. Salt cedar witches'-broom (SCWB), which was extremely pernicious to Salt cedar. It was first observed and reported in Xi'an, China in 2005 (Zhao et al.2005). Witches' broom symptoms were observed on 20 out of 150 (13.3%) salt plants surveyed from the Alar region and 10 out of 86 (11.6%) plants from the Akesu region in southern of Xinjiang in May 2020. The damaged plants compared with asymptomatic plants (Fig.1A), the major symptoms included branches clustered, intersegment shorten and coarsen, giving rise to the formation of clusters (Fig.1B). Total plant DNA was extracted from phloem tissues with asymptomatic symptoms and phloem tissues with witches'-broom symptoms by a CTAB-based DNA extraction method (Green et al.1999). The 16S rRNA gene and the phytoplasma universal primers P1/P7 and rpF1/rpR1 of the rp (ribosomal protein) gene were used for Polymerase chain reaction (PCR) amplification by using the extracted plant total DNA as the template. The PCR product was used as the template and the R16F2n/R16R2 prmer was used for nested PCR amplification of the 16S rRNA gene after the amplification was completed. The results show that no product was obtained in asymptomatic plants. When DNA samples from witches'-broom symptomatic plants were used as templates, fragments with lengths 1219 bp and 1174 bp, corresponding to 16S rRNA gene and rp gene, were obtained. 16S rRNA gene was sequenced and deposited in GenBank under accession number MW447513. BLAST analysis revealed that the partial 16S rRNA sequence of the phytoplasma associated with P. aphylla witches' broom showed highest sequence identity (99.67%) to salt cedar witches' broom phytoplasma, 'Candidatus Phytoplasma tamaricis' (Accession Number: FJ432664). Phylogenetic and molecular evolutionary analyses were conducted using MEGA-X (Kumar et al., 2018). Results showed taht the SCWB and 16S rXXX group's'Candidatus Phytoplasma tamaricis', (GenBank accession: FJ432664) have the highest affinity (Fig.2A). A virtual restriction fragment length polymorphism(RFLP) was done to determinethe subgroup ( Zhao et al. 2009). The 16S rDNA sequence from the Tamarix chinensis plant showed 99.3% similarity with that of the "Candidatus Phytoplasma tamaricis" reference strain (GenBank accession: FJ432664), suggesting that the phytoplasma in this study belongs to "Candidatus Phytoplasma tamaricis"-related strain. Therefore, it can be stated that SCWB belongs to the 16S rXXX group. The partial rp sequences only shared 84.74% sequence similarity with that of 'Candidatus Phytoplasma prunorum' (MG383523) of Apple proliferation group, a known subgroup 16S rX. Blast analysis based on the partial rp sequences showed that it shares less than 90% similarity with that of any known phytoplasma (Fig 2B), we suspect that this is due to a lack of sequenced rp gene sequences for the 16S rXXX group. To our knowledge, this is the first report of Salt Cedar Witches' Broom phytoplasma in Xinjiang province, China. As a consequence, we guess the SCWB phytoplasma rp gene belongs to 16S rXXX-rp group, which is also the first report about the 16SrXXX-rp group. Because SCWB1 is the only strain in the 16S rXXX group, and it is the representative strain of the 16S rXXX-A subgroup (Zhao et al. 2009). So, the SCWB disease we found in southern Xinjiang belongs to the 16S rXXX-A subgroup.

Identification of 16SrII-V Phytoplasma Associated with Mungbean Phyllody Disease in Taiwan.

Mungbean (Vigna radiata (L.) R. Wilczek), an important legume crop in Asia, is primarily cultivated in the central-southern region of western Taiwan. In 2020, mungbean exhibiting typical phytoplasma-induced disease symptoms such as witches' broom, phyllody, virescence, and proliferation was observed in Yunlin County, Taiwan. Moreover, the seed harvested from diseased plants displayed premature germination. Transmission electron microscopy examination of leaf veins prepared from symptomatic mungbean demonstrated that the occlusion of sieve tubes resulted from the accumulation of phytoplasma-like bodies in sieve elements along with filament-like structures in sieve pores. The association of phytoplasma in symptomatic mungbean was confirmed by PCR analyses of the 16S ribosomal RNA (rRNA) and immunodominant membrane protein genes. Further analyses of the 16S rRNA-based phylogenetic tree and the iPhyClassifier-based virtual restriction fragment length polymorphism study demonstrated that the phytoplasma-associated mungbean phyllody disease identified in this study belongs to the 16SrII-V subgroup. BLAST analysis and the phylogenetic analysis indicated that the SAP11-like protein identified in mungbean phyllody disease is identical to peanut witches' broom phytoplasma SAP11, which explains the witches' broom phenotype observed in symptomatic mungbean. The results described in this report confirm that the 16SrII-V phytoplasma, a widely distributed phytoplasma associated with peanut witches' broom disease in Taiwan, has also infected mungbean. This is not only the first instance of mungbean phyllody disease found in Taiwan but also the first instance of mungbean phyllody disease caused by 16SrII-V subgroup phytoplasma.

Molecular identification and characterization of ‘Candidatus Phytoplasma convolvuli’-related strains (representing a new 16SrXII-O subgroup) associated with papaya bunchy top disease in Nigeria

Beginning in 2018, a new papaya disease occurred in Ibadan, Oyo State, Nigeria. Affected papaya trees exhibited symptoms including excessive shoot proliferation at the top of the crown, leaf mosaic and crinkling, and dieback. The disease was designated as Nigerian papaya bunchy top (NGPBT) disease. Since excessive shoot proliferation was the most characteristic symptom of the disease and it was indicative of phytoplasma infection, a polymerase chain reaction (PCR)-based phytoplasma molecular diagnostic method was employed in this study. Phytoplasmas were detected in diseased papaya samples by semi-nested PCR using phytoplasma 16 S rRNA gene-specific primers. Subsequent determination and analysis of the amplified 16 S rRNA gene sequences and phylogenetic analysis revealed that NGPBT phytoplasmas were most closely related to ‘Candidatus Phytoplasma convolvuli’ in the Stolbur (16SrXII) group. Virtual restriction fragment length polymorphism (RFLP) analysis based on the 16 S rRNA gene F2nR2 fragment showed that NGPBT strains represented a novel subgroup in the Stolbur phytoplasma group, designated as 16SrXII-O. This is the first report of a phytoplasma disease in papaya in Nigeria. Identification of the NGPBT strains not only broadened the genetic diversity of the Stolbur phytoplasma group but also signaled an expansion of the phytoplasma group's geographic distribution. Notably, two previously reported phytoplasmas associated with papaya diseases in Australia and Taiwan also belong to the 16SrXII group and exhibit symptoms similar to NGPBT disease. It would be interesting to learn how these three diseases relate to each other. Further in-depth study on weed reservoirs and insect vectors of NGPBT phytoplasmas will contribute to a better understanding of pathosystem and epidemiology of the NGPBT disease.

First report of 'Candidatus Phytoplasma trifolii' associated with leaf reddening and upright growth in pears (Pyrus communis L.)

The natural occurrence of 'Candidatus Phytoplasma trifolii' in pear trees (Pyrus communis Linnaeus) is reported here for the first time. In 2017, a total of thirty-five pear trees, two of them exhibiting leaf rolling along the midvein, reddening, bushy appearance, and upright growth symptoms were sampled in different locations in Van province, Turkey. The total deoxyribonucleic acid was extracted from symptomatic and asymptomatic plants. The purified DNA served as a template in nested polymerase chain reaction (nested-PCR) assays, performed to amplify 16S rRNA sequences using universal primer pairs (R16mF2/R16mR1 and R16F2n/R16R2). The resulting PCR products were then cloned into a pGEM T-Easy vector and sequenced bidirectionally. The phytoplasma strain, group, and subgroup identity were determined using the in silico restriction fragment length polymorphism (RFLP) analysis of the 16S ribosomal RNA-encoding gene sequences profiling with seventeen distinct restriction enzymes. Of the thirty-five pear samples, only two yielded 1 256 bp and 1 258 bp DNA fragments and were designated as Van-Pr3 (Acc. No. MH709141) and Van-Pr4 (Acc. No. MH730561), respectively. Based on the in silico virtual RFLP pattern analysis of the 16S rRNA sequences, we confirmed the presence of 'Ca. P. trifolii' belonging to the clover proliferation group and both identified phytoplasmas were identical with the similarity coefficient of 1.00 to the reference pattern of 16Sr group VI, subgroup A (Acc. No. AY390261). Here we report that the pear tree is an alternate host of the 'Ca. P. trifolii'. 

Identification of Phytoplasmas Representing Multiple New Genetic Lineages from Phloem-Feeding Leafhoppers Highlights the Diversity of Phytoplasmas and Their Potential Vectors.

Phytoplasmas are obligate transkingdom bacterial parasites that infect a variety of plant species and replicate in phloem-feeding insects in the order Hemiptera, mainly leafhoppers (Cicadellidae). The insect capacity in acquisition, transmission, survival, and host range directly determines the epidemiology of phytoplasmas. However, due to the difficulty of insect sampling and the lack of follow-up transmission trials, the confirmed phytoplasma insect hosts are still limited compared with the identified plant hosts. Recently, quantitative polymerase chain reaction (qPCR)-based quick screening of 227 leafhoppers collected in natural habitats unveiled the presence of previously unknown phytoplasmas in six samples. In the present study, 76 leafhoppers, including the six prescreened positive samples, were further examined to identify and characterize the phytoplasma strains by semi-nested PCR. A total of ten phytoplasma strains were identified in leafhoppers from four countries including South Africa, Kyrgyzstan, Australia, and China. Based on virtual restriction fragment length polymorphism (RFLP) analysis, these ten phytoplasma strains were classified into four distinct ribosomal (16Sr) groups (16SrI, 16SrIII, 16SrXIV, and 16SrXV), representing five new subgroups (16SrI-AO, 16SrXIV-D, 16SrXIV-E, 16SrXIV-F, and 16SrXV-C). The results strongly suggest that the newly identified phytoplasma strains not only represent new genetic subgroup lineages, but also extend previously undiscovered geographical distributions. In addition, ten phytoplasma-harboring leafhoppers belonged to seven known leafhopper species, none of which were previously reported insect vectors of phytoplasmas. The findings from this study provide fresh insight into genetic diversity, geographical distribution, and insect host range of phytoplasmas. Further transmission trials and screening of new potential host plants and weed reservoirs in areas adjacent to collection sites of phytoplasma harboring leafhoppers will contribute to a better understanding of phytoplasma transmission and epidemiology.

Molecular identification of 16srii-d phytoplasmas infecting Crossandra infundibuliformis in India

The symptoms of little leaf, leaf curling, malformed inflorescences, yellowing, and stunting were observed in Crossandra infundibuliformis plants during a field inspection in Mysuru district, Karnataka state, India. The disease incidence ranged from 0.2 to 4%. The DNA extracted from the leaf midrib tissues of symptomatic samples was amplified in polymerase chain reaction assays using 16S rRNA and protein translocase secA genes primers confirming the presence of these bacteria in the symptomatic plants. The nucleotide sequence analysis and the phylogenetic comparison indicated the presence of a ‘Candidatus Phytoplasma australasia’-related strain. The virtual restriction fragment length polymorphism analysis showed that the phytoplasmas associated with the infected crossandra samples belonged to the 16SrII-D subgroup. This report describes the first identification of this phytoplasma strain in symptomatic crossandra plants in India.

Detection and Molecular Characterization of ‘Candidatus Phytoplasma Trifolii’, a Member of the Clover Proliferation Grup, Infecting Tomato Plants from Iğdır Province in Turkey

The tomato plant exhibiting leaf rolling, witches’ broom, distorted and elongated flower’s sepals in Iğdır province, Turkey, was observed. Total DNA extraction was performed from the symtomatic fresh tomato sample. All DNAs were subjected to Direct and Nested polymerase chain reaction (PCR) with universal primer sets that amplified the 16S rRNA of phytoplasmas. PCR products were purified from agarose gel and cloned into the pGEM T-Easy cloning vector. Recombinant plasmids were introduced into the prokaryotic cloning bacteria by electroporation. Plasmid isolation was performed by selecting one of the positive clones randomly and sequencing was performed by Next Generation Sequencing (NGS). Sequencing results revealed that the 16S rRNA gene associated with phytoplasma was 1251 nucleotides in size, and this sequence was denominated as ‘Iğdır 10’ isolates and recorded in the GenBank under the MT344968 accession number. The virtual restriction fragment length polymorphism (V-RFLP) and phylogenetic analysis of the 16S rDNA sequence confirmed that the cause of disease in infected tomato plants was 'Candidatus Phytoplasma trifolii' ('Ca. P. trifolii') (16SrVI-A, Clover proliferation group), with a 1.00 similarity coefficient. This present study is the first report of ‘Ca. P. trifolii’ and its nucleotide sequence analysis in naturally infected tomato in Iğdır province.