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During field surveys conducted in 2021 in western Jordanian heights, phytoplasma-like symptoms were observed, including leaf reddening along with slow decline in pear (pear decline symptoms) and leaf yellowing followed by scorch in apple trees. Abundant populations of the psyllid Cacopsylla bidens were found in the pear trees. Nested PCR amplifying 16S rDNA detected phytoplasmas in 63% and 36% of symptomatic pear and apple samples, respectively, and in 46% of pooled C. bidens specimens. No phytoplasmas were detected in symptomless plant samples. PCR product nucleotide sequence analyses attributed the phytoplasmas detected in pear to ‘Candidatus Phytoplasma pyri’, ‘Ca. P. solani’, ‘Ca. P. omanense’, and ‘Ca. P. aurantifolia’, and those identified in apple to ‘Ca. P. solani’ and ‘Ca. P. omanense’. All phytoplasma strains identified in C. bidens were attributed to ‘Ca. P. pyri’. To the best of our knowledge, this is the first report of phytoplasmas associated with pear and apple diseases, including pear decline, in Jordan. Even though a limited number of pome fruit samples were analyzed, a surprising diversity was found among detected phytoplasmas. Further studies will be carried out to investigate the complex etiology of such diseases, and the ‘Ca. P. pyri’ vectoring activity of C. bidens.

Phytoplasmas are bacteria transmitted by insects that can cause plant diseases. In Bogotá ' Candidatus Phytoplasma asteris' and ' Candidatus Phytoplasma fraxini', infect 11 species of urban trees, weeds, grass, potato and strawberry. A set of primers, that amplify both phytoplasmas species were designed and used for absolute and relative qPCR quantification of the 16SrRNA gene. The primers AJ-16Sr-F/AJ-16Sr-R allowed the amplification of ‘ Ca. P. asteris’, ‘ Candidatus Phytoplasma palmae’, ‘ Ca. P. fraxini’ and ‘ Candidatus Phytoplasma phoenicium’, not of ‘ Candidatus Phytoplasma pruni’. Absolute qPCR detected phytoplasmas between 1 × 10 9 and 1 × 10 3 copies/μL DNA extract. Two species-specific hydrolysis probes, AJ-16SrI-Cy5.5 and AJ-16SrVII-TexRed, were designed to detect ' Ca . P. asteris' and ' Ca . P. fraxini' respectively, using the AJ-16Sr-F/AJ-16Sr-R primers. For relative quantification, the 18SrRNA gene was used as normalizer. Relative qPCR detected phytoplasmas between 1 × 10 9 and 1 × 10 3 copies/μL DNA extract. Multiplex reactions allowed the specific quantification of ' Ca . P. asteris', ' Ca . P. fraxini' in comparison to the normalizer. qPCR methods were validated on natural hosts Andean oak trees and leafhoppers . The relative quantification values were higher for ' Ca . P. fraxini' (x̅ RQ = 3203.1 ± 2622,9 n  = 14) compared with ' Ca . P. asteris' (x̅ RQ = 14.9 ± 24,5 n  = 6) in oak tree samples. In the leafhoppers, the relative quantification values ranged between RQ = 26.5 and RQ = 294,927.3 for ' Ca . P. fraxini’ and RQ = 34.8 and RQ = 1722.2 for ' Ca . P. asteris'. In conclusion, although absolute qPCR allowed the quantification of phytoplasmas by comparing Cq (quantification cycle) values of samples with a standard curve, it did not allow to differentiate between ' Ca . P. asteris' and ' Ca. P. fraxini'. In contrast, relative qPCR assays using specific hydrolysis probes allowed the specific detection and quantification of each phytoplasma, in individual and mixed infections in insect vectors and plant hosts.

Numerous yellows-type diseases of plants have been associated with wall-less prokaryote pathogens – phytoplasmas over the last 40 years. These pathogens are not grown in axenic culture till now so that advances in their study are mainly achieved by molecular techniques. Severe disease epidemics associated with phytoplasma presence have been described worldwide. These include coconut lethal yellowing in Africa and the Caribbean, grapevine yellows in major viticultural areas and various diseases affecting stone and pome fruit plants. Phytoplasma-infected plants exhibit symptoms suggesting a profound disturbance in the normal balance of growth regulators and also yellows symptoms, but very often the symptomatology is not diagnostic. Detection and characterization of phytoplasmas infecting different plant species are now possible with molecular methods, based on the study of 16S rDNA polymorphisms. Molecular diversity of phytoplasmas is also demonstrated by studying genes coding the ribosomal proteins S3, tuf, SecY, amp, imp and other genes. Four phytoplasma genomes have been fully sequenced, including those of two 'Candidatus Phytoplasma asteris' strains, and those of strains of 'Ca. P. mali' and 'Ca. P. australiense'. Three of these genomes contain large amounts of repeated DNA sequence, and the fourth carries multiple copies of almost 100 genes. Considering that phytoplasmas have unusually small genomes, these repeats might be related to their transkingdom habitat and to their pathogenic activity. An outlook of recent findings in the field is also reported.

Symptoms of decline, leaf yellowing and reddening, little leaf and malformation were observed in apricot, guava, lychee, mango, pomegranate and grapevine in fruit orchards belonging to three states of India namely Delhi, Maharashtra and Jammu & Kashmir during 2016–2017. Phytoplasmas belonging to three different groups were detected in all the symptomatic fruit tree samples in polymerase chain reaction with phytoplasma specific primer pairs amplifying 16S rRNA and secA genes. Pair wise sequence comparison and phylogenetic analysis confirmed the presence of phytoplasmas. A ‘Candidatus Phytoplasma asteris’ - related strain was detected in apricot showing decline symptoms in Siot (Jammu & Kashmir). ‘Ca. P. australasia’ - related strain was identified in guava, lychee, mango and pomegranate in Rajbag, Sunjwan (Jammu & Kashmir), Pusa (Delhi) and Baramati (Maharashtra), respectively, exhibiting little leaf, leaf yellows and malformation symptoms. Furthermore, a rice yellow dwarf group (16SrXI-B) - related strain was detected in pomegranate and grapevine that showed leaf yellowing and reddening at Pusa (Delhi) and Baramati (Maharashtra), respectively. Subgroup analysis using virtual RFLP of 16S rDNA sequences allowed enclosing these phytoplasma strains into 16SrI-B, 16SrII-D and 16SrXI-B subgroups. In the study, identification of phytoplasma subgroups, 16SrI-B in apricot, 16SrII-D in guava, lychee, mango and pomegranate (Pusa) and 16SrXI-B in pomegranate (Baramati) and grapevine, are the first records globally.

Cassava witches’ broom disease (CWBD) is one of the most devastating diseases of cassava (Manihot esculenta Crantz), and it threatens global production of the crop. In 2017, a phytoplasma, Candidatus Phytoplasma luffae (Ca. P. luffae), was reported in the Philippines, and it has been considered as the causal agent, despite unknown etiology and transmission of CWBD. In this study, the nationwide occurrence of CWBD was assessed, and detection of CWBD’s pathogen was attempted using polymerase chain reaction (PCR) and next-generation sequencing (NGS) techniques. The results showed that CWBD has spread and become severe, exhibiting symptoms such as small leaf proliferation, shortened internodes, and vascular necrosis. PCR analysis revealed a low phytoplasma detection rate, possibly due to low titer, uneven distribution, or absence in the CWBD-symptomatic cassava. In addition, NGS techniques confirm the PCR results, revealing the absence or extremely low phytoplasma read counts, but a surprisingly high abundance of fastidious and xylem-limited fungus, Ceratobasidium sp. in CWBD-symptomatic plants. These findings cast doubt over the involvement of phytoplasma in CWBD and instead highlight the potential association of Ceratobasidium sp., strongly supporting the recent findings in mainland Southeast Asia. Further investigations are needed to verify the etiology of CWBD and identify infection mechanisms of Ceratobasidium sp. to develop effective diagnostic and control methods for disease management.

The phytoplasma associated diseases are an emerging threat to fruit and vegetable crops leading severe yield losses worldwide. Pear (Pyrus communis L.) trees, with symptoms of severe reddening, dwarfing and shoot proliferation were observed in pear orchards of Malatya province of Turkey. Tomato (Solanum lycopersicum L.) plants grown nearby the symptomatic pear orchard displaying leaf rolling, severe flower sterility and purple leaves were observed at the same agroecosystem. To verify the presence and diversity of phytoplasmas, symptomatic pears and tomatoes were sampled and weeds nearby the symptomatic plants were collected. Total plant DNA was purified from midrib of collected leaves using a commercial kit. The DNA samples were analyzed by nested polymerase chain reaction (PCR) using universal primer pairs to amplify 16S rDNA fragments. The phytoplasmas detected in collected samples were differed according to the host. Here we detected and characterized ‘Candidatus Phytoplasma pyri’ belonging to apple proliferation group (subgroup 16SrX-C) from a pear tree, ‘Candidatus Phytoplasma trifolii’ belonging to clover proliferation group (subgroup 16SrVI-A) from a weed (Amaranthus retroflexus) and ‘Candidatus Phytoplasma solani’ belonging to the stolbur phytoplasma group (subgroup 16SrXII-A) from a tomato plant. Direct sequencing of PCR products verified the phytoplasmal nature of the infections. The occurrence of ‘Ca. P. trifolii’ on A. retroflexus is the first report for the world. The irregular presence of the phytoplasmas in fruit and vegetable crops and weeds indicates continuous spread of the phytoplasmas threatening the new crops and new horizons.

Phytoplasmas from the 16Sr-X apple proliferation (AP) group are quarantine species in Europe and causal agents of the most important diseases of fruit trees within the family Rosaceae, namely apple proliferation, European stone fruit yellows and pear decline. In this study, a detailed insight into the molecular diversity of isolates of two phytoplasmas from the AP group, i. e. ‘Candidatus Phytoplasma mali’ and ‘Ca. P. prunorum’ obtained from different orchards in Slovenia, was estimated by a multilocus sequence typing, based on analysis of the genomic regions of aceF, pnp, secY and imp. With seven and five genotypes defined for ‘Ca. P. mali’ and ‘Ca. P. prunorum’ isolates, respectively, imp was the most variable among the applied markers. On the other hand, pnp was the least variable with three genotypes defined for ‘Ca. P. mali’ isolates and only one for ‘Ca. P. prunorum’ isolates. The presented results complete the survey of the AP group phytoplasma diversity in Slovenia, which has started with the recent analysis of the ‘Ca. P. pyri’. The comparison of results with those from several European countries shows an important genetic diversity of the Slovenian genotypes with some previously unknown. The genotype distribution reflects the geographic position of Slovenia. Additional grafting experiments with apricot trees tolerant to ‘Ca. P. prunorum’ demonstrated that the tolerance status is transmissible. Some possible mechanisms involved in the process are discussed.

The lowest disease incidence was 1% for the Dakashina Kannada and Tumkur districts in Karnataka.The most prominent symptom in infected palms was short, dark green leaves with a wavy leaf lamina. Areca palm with crown choking disease symptoms: black, crinkled and brittle roots (left) and pinkish / brownish discoloration of the phloem (right) Seven leaf samples were collected from diseased palms growing in the Zonal Agricultural & Horticultural Research Station, Keladi Shivappa Nayaka University of Agricultural and Horticultural Sciences in Shivamogga, Karnataka (Latitude: 13° 55' 47.748“. Typical phytoplasma pleomorphic bodies lacking cell walls were seen in the sieve tube cells of leaf midrib sections from diseased areca palms, when examined by transmission electronic microscopy (Spurr, 1969) at the Sri Sai Histology Center, Hyderbad India (Fig. 4).

Phyllody associated with the presence of ‘ Candidatus Phytoplasma’ strains is the most important disease, severely affecting sesame plants, either partially or completely. Seven species of sesame, namely Sesamum alatum , S. indicum , S. malabaricum , S. mulayanum , S. radiatum , S. laciniatum and S. prostratum were studied for their resistance or susceptibility to phyllody, symptomatology and identification of the phytoplasma involved in symptom development. Symptoms viz ., phyllody and flower virescence were recorded in all the seven species of Sesamum with varying degrees of susceptibility. Some sesame species exhibited yellowing of leaves, stunted growth, little leaves, proliferation of axillary shoots, witches’-broom, and splitting of capsules. The lowest incidence of phyllody was found in S. alatum (0–6%), followed by S. laciniatum (11–17%) and S. prostratum (16–27%). The incidence of phyllody in S. indicum, S. malabaricum , S. mulayanum and S. radiatum was between 44–68%. In the present investigation, two phytoplasma strains, 16SrI-B (‘ Candidatus Phytoplasma asteris’) and 16SrII-D (‘ Candidatus Phytoplasma australasia’) were identified in phyllody affected Sesamum species. The perennial species S. laciniatum and S. prostratum , found only in India, are at risk of extinction due to phyllody and pose a serious threat as inoculum source for the spread of the disease. In the future, focus on genome sequencing of ‘ Candidatus Phytoplasma’ strains associated with phyllody in Sesamum species and identification of the insect vectors involved in phytoplasma transmission are essential to clarify the phytoplasma strain identity and understand the host–pathogen-vector relationship.

Grapevine yellows (GY) affecting Lebanese vineyards are reported to be associated with ‘Candidatus Phytoplasma solani’, the bois noir (BN) etiological agent. However, during a field survey in June 2014 for BN in Mansoura municipality of West Bekaa, Lebanon, ‘Candidatus Phytoplasma omanense’ was detected in a grapevine sample, cultivar Syrah, exhibiting leaf scorch and discoloration, using a phytoplasma universal nested-PCR and sequencing of the 1.2 kbp 16SrDNA amplicon. The same 1.2 kbp 16SrDNA sequence could be amplified from Hyalesthes obsoletus and Reptalus sp. Cixiidae planthoppers collected on Convolvulus arvensis (bindweed) plants in June 2014 in Aammiq municipality of West Bekaa. Yellowing and stunted bindweed plants collected in 2015 in Kefraya and Aamiq municipalities of West Bekaa were also found infected with the same phytoplasma strain. A 16S rDNA RFLP assay was designed to differentiate this phytoplasma from ‘Ca. P. solani’ and ‘Ca. P. phoenicium’, which are endemic to the Bekaa region. ‘Ca. P. omanense’ is reported for the first time in grapevine and in Cixiidae planthoppers already known to respectively host and vector ‘Ca. P. solani’ strains associated with BN disease in grapevine. This result highlights the need for a precise survey of phytoplasmas associated to grapevine yellows in the Eastern Mediterranean basin, and for investigating the possible role of ‘Ca. P. omanense’ as a new threat to Euro-Mediterranean viticulture.