Distinct Phytoplasma Research Articles

A metagenomic investigation of phytoplasma diversity in Australian vegetable growing regions.

In this study, metagenomic sequence data was used to investigate the phytoplasma taxonomic diversity in vegetable-growing regions across Australia. Metagenomic sequencing was performed on 195 phytoplasma-positive samples, originating either from historic collections (n=46) or during collection efforts between January 2015 and June 2022 (n=149). The sampled hosts were classified as crop (n=155), weed (n=24), ornamental (n=7), native plant (n=6), and insect (n=3) species. Most samples came from Queensland (n=78), followed by Western Australia (n=46), the Northern Territory (n=32), New South Wales (n=17), and Victoria (n=10). Of the 195 draft phytoplasma genomes, 178 met our genome criteria for comparison using an average nucleotide identity approach. Ten distinct phytoplasma species were identified and could be classified within the 16SrII, 16SrXII (PCR only), 16SrXXV, and 16SrXXXVIII phytoplasma groups, which have all previously been recorded in Australia. The most commonly detected phytoplasma taxa in this study were species and subspecies classified within the 16SrII group (n=153), followed by strains within the 16SrXXXVIII group ('Ca. Phytoplasma stylosanthis'; n=6). Several geographic- and host-range expansions were reported, as well as mixed phytoplasma infections of 16SrII taxa and 'Ca. Phytoplasma stylosanthis'. Additionally, six previously unrecorded 16SrII taxa were identified, including five putative subspecies of 'Ca. Phytoplasma australasiaticum' and a new putative 16SrII species. PCR and sequencing of the 16S rRNA gene was a suitable triage tool for preliminary phytoplasma detection. Metagenomic sequencing, however, allowed for higher-resolution identification of the phytoplasmas, including mixed infections, than was afforded by only direct Sanger sequencing of the 16S rRNA gene. Since the metagenomic approach theoretically obtains sequences of all organisms in a sample, this approach was useful to confirm the host family, genus, and/or species. In addition to improving our understanding of the phytoplasma species that affect crop production in Australia, the study also significantly expands the genomic sequence data available in public sequence repositories to contribute to phytoplasma molecular epidemiology studies, revision of taxonomy, and improved diagnostics.

Identification of phytoplasmas associated with grapevine ‘bois noir’ and flavescence dorée in inter-row groundcover vegetation used for green manure in Franciacorta vineyards

‘Bois noir’ (BN) and flavescence dorèe (FD), the two main diseases of the grapevine yellows complex associated with genetically distinct phytoplasmas, have a complex epidemiology including multiple insect vectors and reservoir plants. This study investigated the presence of BN and FD phytoplasmas in nine groundcover plant species commonly utilized for inter-row vineyard green manure in Franciacorta (North Italy). The activities conducted in 2020 included monitoring and sampling groundcover plant species and symptomatic grapevines in September, and phytoplasma identification and typing by amplification and sequence analyses of stamp and map genes. Molecular analyses identified BN phytoplasma (strains carrying the stamp gene sequence variants St5, St19, St30) and FD phytoplasma (strains carrying the map gene sequence variant M54) in 72% and 28% of symptomatic grapevines, respectively. BN phytoplasma strains St5 and St30 were found also in Eruca sativa, Vicia sativa, and Polygonum fagopyrum. FD phytoplasma strain M54 was found also in Vicia faba, Trifolium incarnatum, and Polygonum fagopyrum. These results reinforced the evidence of the increasing range of BN and FD phytoplasma alternative plant hosts and suggested a criterium for the selection of the groundcover plant species utilized for green manure, excluding the ones putatively involved in BN and FD diffusion.

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.

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.

Tri-Trophic Relationship among Sugarcane White Leaf Disease Phytoplasma (WLDP), <em>Deltocephalus menoni</em> (Homoptera: Cicadellidae) and Sugarcane Plant in Secondary Transmission of the Disease

Purpose: Sugarcane White Leaf Disease (WLD) is one of the most devastative diseases in cane-sugar industry in Sri Lanka and the causative agent is a distinct phytoplasma (WLDP) strain within the 16SrXI. White Leaf Disease spreads through infected seed cane and insect vector; Deltocephalus menoni (Hemiptera: Cicadellidae). This paper presents the tri-trophic relationship of the plant, vector and pathogen in disease transmission. Research Method: Four-month-old sugarcane plants of the variety SL 96 128 and laboratory-reared populations of D. menoni were used for this study. Performance of D. menoni on healthy plants and WLD plants was examined in relation to the aggregation, amount of feeding, fecundity, number of nymphs emerged, nymphal period, survival of nymph and adult’s longevity. The plant architecture, leaf histological and physiological aspects were also studied. Findings: Except for nymphal period, all behavioural characteristics were significantly-high in WLDinfected plants in 10, 14, 30, 8, 12 and 17 percent, respectively. The nymphal period of D. menoni was significantly-reduced by 12.78 percent. Number of shoots (41.83 percent) and leaves (18.96 percent) were high and distance between two leaves tends to be reduced by 32.85 percent in diseased-plants altering plant architecture into a profuse and bushy nature. Sucking distance, thicknesses of lower epidermis and phloem fibre layer in leaves were significantly reduced in infected plants. Leaf N, P, K and moisture content increased (24.89, 34.78, 28.64 and 3.85percent) while leaf wax content was reduced by 4.93 percent. Originality/value: Disease infected plants are augmenting its vector; D. menoni by attracting, enhancing survival and population build-up which promotes the transmission of WLD phytoplasma itself. There by regular inspection and rogueing out of WLD infected clumps make a great impact on secondary transmission of WLD by D. menoni.

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.

Detection and molecular characterization of two genetically distinct phytoplasmas associated with mungbean seed pod abortion in Iran

New symptoms including aborted seed pods disease were observed on mungbean (Vigna radiata) plants in the most important growing areas in Iran that was similar to symptoms of phytoplasma disease previously reported from Australia. Thirty samples were collected from symptomatic and non-symptomatic plants, followed by PCR amplification, DNA sequencing, RFLP and phylogenetic analysis of 16S ribosomal RNA gene to detect and identify possible associated phytoplasmas. Results indicated that two different phytoplasmas, related to ‘Candidatus Phytoplasma trifolii’ and ‘Candidatus Phytoplasma aurantifolia’, were associated with different symptomatic plants samples from the same field, mixed infection was not observed in these samples. The sequence analysis results indicated that the nucleotide sequences from mungbean isolates 47 (MT674290), 7 (MT674291) and 32 (MT674292) had 98.8% sequence identity to ‘Candidatus Phytoplasma aurantifolia’ isolate TBBP-H1 (Acc. No: MN565885), while isolates 80 (MT674293) and 25 (MT674294) had 99.8% sequence identity similar to ‘Candidatus Phytoplasma trifolii’ (MF092789). In silico RFLP and phylogenetic analyse of mungbean seed pod abortion (MubSpa) phytoplasma indicated that the associated phytoplasmas belonged to phytoplasmas of subgroups 16SrVI-A and 16SrII-D. To the best of our knowledge, this is the first report of association ‘Ca. P. trifolii’ and ‘Ca. P. aurantifolia’ strains infecting mungbean plants in Iran.

'Candidatus Phytoplasma stylosanthis', a novel taxon with a diverse host range in Australia, characterised using multilocus sequence analysis of 16S rRNA, secA, tuf, and rp genes.

In Australia, Stylosanthes little leaf (StLL) phytoplasma has been detected in Stylosanthes scabra Vogel, Arachis pintoi Krapov, Saccharum officinarum L., Carica papaya L., Medicago sativa L., and Solanum tuberosum L. The 16S rRNA gene sequence of StLL phytoplasma strains from S. scabra, C. papaya, S. officinarum and S. tuberosum were compared and share 99.93–100 % nucleotide sequence identity. Phylogenetic comparisons between the 16S rRNA genes of StLL phytoplasma and other ‘Candidatus Phytoplasma’ species indicate that StLL represents a distinct phytoplasma lineage. It shares its most recent known ancestry with ‘Ca. Phytoplasma luffae’ (16SrVIII-A), with which it has 97.17–97.25 % nucleotide identity. In silico RFLP analysis of the 16S rRNA amplicon using iPhyClassifier indicate that StLL phytoplasmas have a unique pattern (similarity coefficient below 0.85) that is most similar to that of ‘Ca. Phytoplasma luffae’. The unique in silico RFLP patterns were confirmed in vitro. Nucleotide sequences of genes that are more variable than the 16S rRNA gene, namely tuf (tu-elongation factor), secA (partial translocation gene), and the partial ribosomal protein (rp) gene operon (rps19-rpl22-rps3), produced phylogenetic trees with similar branching patterns to the 16S rRNA gene tree. Sequence comparisons between the StLL 16S rRNA spacer region confirmed previous reports of rrn interoperon sequence heterogeneity for StLL, where the spacer region of rrnB encodes a complete tRNA-Isoleucine gene and the rrnA spacer region does not. Together these results suggest that the Australian phytoplasma, StLL, is unique according to the International Organization for Mycoplasmology (IRPCM) recommendations. The novel taxon ‘Ca. Phytoplasma stylosanthis’ is proposed, with the most recent strain from a potato crop in Victoria, Australia, serving as the reference strain (deposited in the Victorian Plant Pathology Herbarium as VPRI 43683).

First detection of “Candidatus Phytoplasma asteris” ‐ and “Candidatus Phytoplasma solani”‐related strains in fig trees

AbstractIn July 2017, a survey was conducted in a fig collection plot at Locorotondo (south of Italy) to investigate the possible presence of phytoplasmas in plants showing yellowing, deformed leaves, short internodes, mottling and mosaic. Samples were collected from symptomatic plants and tested by nested PCR assays using universal and specific primers to amplify the 16S rDNA of these prokaryotes. PCR results detected the presence of phytoplasma sequences in twenty plant samples that resulted clustering two phylogenetically distinct phytoplasmas, i.e., “Candidatus Phytoplasma asteris” and “Candidatus Phytoplasma solani” affiliated to 16SrI and 16SrXII ribosomal groups, respectively. The presence of phytoplasmas belonging to both ribosomal groups was confirmed with group specific quantitative PCR and RFLP assays on 16S ribosomal amplicons. Results of this study indicate for the first time the occurrence of phytoplasmas in fig; however, more work should be carried out to verify their association with the symptoms observed on diseased fig plants.

Molecular study of two distinct phytoplasma species associated with streak yellows of date palm in Iran

AbstractA disease with symptoms similar to palm lethal yellowing was noticed in the early 2013 in Khuzestan Province (Iran) in date palm (Phoenix dactylifera). Infected trees displaying symptoms of streak yellows and varied in the incidence and severity of yellowing. A study was initiated to determine whether phytoplasma was the causal agent. Polymerase chain reaction–restriction fragment length polymorphism (PCR‐RFLP) methods using universal phytoplasma primers pairs R16mF1/mR1 and M1/M2 were employed to detect putative phytoplasma(s) associated with date palm trees. Nested PCR using universal primers revealed that 40 out of 53 trees were positive for phytoplasma while asymptomatic date palms from another location (controls) tested negative. RFLP analyses and DNA sequencing of 16S rDNA indicated that the presence of two different phytoplasmas most closely related to clover proliferation (CP) phytoplasma (group 16SrVI) and ash yellows (AY) phytoplasma (group 16SrVII). Sequence analysis confirmed that palm streak yellows phytoplasmas in each group were uniform and to be phylogenetically closest to “CandidatusP. fraxini” (MF374755) and “Ca. P. trifolii” isolate Rus‐CP361Fc1 (KX773529). Result of RFLP analysis of secA gene of positive samples using TruI and TaqI endonuclease is in agreement with rDNA analysis. On this basis, both strains were classified as members of subgroups 16SrVI‐A and 16SrVII‐A. This is the first report of a phytoplasma related to CP and AY phytoplasma causing date palm yellows disease symptoms.

PCR-Based Sequence Analysis on Multiple Genes Other than 16S rRNA Gene for Differentiation of Phytoplasmas.

Differentiation and classification of phytoplasmas have been primarily based on the highly conserved 16S rRNA gene, for which "universal" primers are available. To date, 36 ribosomal (16Sr) groups and more than 150 subgroups have been delineated by RFLP analysis of 16S rRNA gene sequences. However, in recent years, the use of moderately conserved genes as additional genetic markers has enhanced the resolving power in delineating distinct phytoplasma strains among members of some 16Sr subgroups.This chapter describes the methodology of amplification, differentiation, and classification of phytoplasma based on less-conserved non-ribosomal genes, named rp and secY. Actual and virtual RFLP analyses of amplicons obtained by semi-universal or group-specific rp and secY gene-based primers are used for finer differentiation of phytoplasma strains within a given group. The rp and secY gene-based classification not only readily resolves 16Sr subgroups within a given 16Sr group, but also provides finer differentiation of closely related phytoplasma strains within a given 16Sr subgroup.

'Candidatus Phytoplasma wodyetiae', a new taxon associated with yellow decline disease of foxtail palm (Wodyetia bifurcata) in Malaysia.

Landscape-grown foxtail palm (Wodyetia bifurcata A. K. Irvine) trees displaying symptoms of severe foliar chlorosis, stunting, general decline and mortality reminiscent of coconut yellow decline disease were observed in Bangi, Malaysia, during 2012. DNA samples from foliage tissues of 15 symptomatic palms were analysed by employing a nested PCR assay primed by phytoplasma universal ribosomal RNA operon primer pairs, P1/P7 followed by R16F2n/R2. The assay yielded amplicons of a single band of 1.25 kb from DNA samples of 11 symptomatic palms. Results from cloning and sequence analysis of the PCR-amplified 16S rRNA gene segments revealed that, in three palms, three mutually distinct phytoplasmas comprising strains related to 'Candidatus Phytoplasma asteris' and 'Candidatus Phytoplasma cynodontis', as well as a novel phytoplasma, were present as triple infections. The 16S rRNA gene sequence derived from the novel phytoplasma shared less than 96 % nucleotide sequence identity with that of each previously describedspecies of the provisional genus 'Ca. Phytoplasma', justifying its recognition as the reference strain of a new taxon, 'Candidatus Phytoplasma wodyetiae'. Virtual RFLP profiles of the R16F2n/R2 portion of the 16S rRNA gene and the pattern similarity coefficient value (0.74) supported the delineation of 'Ca. Phytoplasma wodyetiae' as the sole representative subgroup A member of a new phytoplasma ribosomal group, 16SrXXXVI.

Expanding and exploring the diversity of phytoplasmas from lucerne (Medicago sativa).

Phytoplasmas are a group of insect-vectored bacteria responsible for disease in many plant species worldwide. Among the crop species affected is the economically valuable forage species lucerne. Here we provide comprehensive molecular evidence for infection in multiple lucerne plants by a phytoplasma not previously known from this plant species. This phytoplasma had a >99% genetic similarity to an unclassified 16S rRNA subgroup previously reported as Stylosanthes little leaf from Stylosanthes spp. and was genetically and symptomatically distinct from a co-occurring but less common 16SrIIA group phytoplasma. Neighbour-joining analyses with publicly available sequence data confirmed the presence of two distinct phytoplasma lineages in the plant population. No PCR detections were made among 38 individuals of 12 co-occurring weed species. Sequence analysis revealed that all nine PCR detections from among 106 individuals of five Hemiptera insect species from the site, three of which had previously been reported as likely vectors, were false positives. This study demonstrates the importance of sequencing to complement PCR detection and avoid potentially inaccurate conclusions regarding vectors, highlights that sampling over a wide spatio-temporal scale is important for vector and alternative host studies, and extends to eight the number of phytoplasma 16 Sr groups known from lucerne.

Identification and Characterization of New 'Candidatus Phytoplasma solani' Strains Associated with Bois Noir Disease in Vitis vinifera L. Cultivars Showing a Range of Symptom Severity in Georgia, the Caucasus Region.

Evidence from a preliminary survey highlighted that 'Candidatus Phytoplasma solani', the etiological agent of bois noir (BN) disease of grapevine, infects grapevine varieties in Georgia, a country of the South Caucasus. In this study, field surveys were carried out to investigate the BN symptom severity in international and Georgian native varieties. 'Ca. P. solani' was detected and identified by polymerase chain reaction-based amplification and restriction fragment length polymorphism analysis of 16S ribosomal DNA, and further characterized by multiple gene typing analysis (vmp1 and stamp genes). Obtained data highlighted that the majority of Georgian grapevine varieties showed moderate and mild symptoms, whereas international cultivars exhibited severe symptoms. Molecular characterization of 'Ca. P. solani' from grapevine revealed the presence of 11 distinct phytoplasma types. Only one type (VmGe12/StGe7) was identical to a strain previously reported in periwinkle from Lebanon; the other 'Ca. P. solani' types are described here for the first time. Phylogenetic analyses of vmp1 and stamp gene concatenated nucleotide sequences showed that 'Ca. P. solani' strains in Georgia are associated mainly with the bindweed-related BN host system. Moreover, the fact that 'Ca. P. solani' strains are distributed in grapevine cultivars showing a range of symptom intensity suggests a different susceptibility of such local cultivars to BN.

CURRENT STATUS OF PHYTOPLASMA DISEASES OF PEACH

Peach (Prunus persica) is in many countries severely affected by yellows and decline diseases of phytoplasmal etiology. These diseases are caused by phylogenetically different phytoplasmas. X-disease is one of the most serious diseases of peach. It is widespread in North America and has not been observed elsewhere. This disease is induced by a distinct phytoplasma, the X-disease agent ‘Candidatus Phytoplasma pruni’, which is a member of the X-disease phytoplasma group or 16SrIII group, subgroup 16SrIII-A. ‘Ca. P. pruni’ is, at the level of 16S rDNA sequences, a homogeneous pathogen. Peach rosette, peach red suture, and peach yellows, also called little peach, are three other diseases which are mainly known from eastern North America. These diseases are currently of minor importance. They are also caused by the X-disease phytoplasma. Peach yellow leaf roll (PYLR) is a major disease of peach that occurs in California. The causative agent, the PYLR phytoplasma, is a member of the apple proliferation (AP) group and is most closely related to the pear decline phytoplasma. In Europe, peach is affected by an economically important disorder known as European stone fruit yellows. This disorder is induced by ‘Ca. P. prunorum’ which is a member of the AP group, subgroup 16SrX-B. ‘Ca. P. prunorum’ is closely related to other important temperate fruit tree phytoplasmas including the PYLR agent. In Lebanon and Iran, peach is affected by almond witches’-broom, a lethal disease that is induced by ‘Ca. P. phoenicium’. This phytoplasma is a member of the 16SrIX group, subgroup 16SrIX-B. Phytoplasmas of other phylogenetic groups, which are known to infect a wide range of plant hosts, have been identified in declining peach trees in several fruit growing areas worldwide. However, the pathological relevance of these non-peach phytoplasmas needs to be investigated in many cases.

Bougainvillea Shoot Proliferation, a New Disease Induced by Distinct Phytoplasmas

AbstractBougainvillea‐potted plants exhibiting typical phytoplasma‐induced symptoms, characterized by foliar chlorosis, shoot proliferation, leaf and bract deformations, and decline were observed in commercial nurseries, located in the state of São Paulo, Brazil. In this study, PCR assays using group‐specific primers revealed that phytoplasmas affiliated with the groups 16SrI and 16SrIII were associated with symptomatic plants. Molecular analysis based on conventional and virtual RFLP patterns and similarity coefficient calculations identified these phytoplasmas as belonging to subgroups 16SrI‐B and 16SrIII‐B. Phylogenetic analysis confirmed that these phytoplasmas were closely related to representatives of both subgroups. Transmission assays using dodder supported the initial evidence that the symptoms were associated with phytoplasmas.

First Report of a 16Sr I-B Phytoplasma Associated with Phyllody and Stem Fasciation of Flax (Linum usitatissimum) in India.

Flax or linseed is grown as a fiber or oilseed crop in tropical and temperate regions. It is commercially cultivated in many countries of the world including Canada, China, India, the United States, Ethiopia, Pakistan, Russia, Poland, and Argentina (1). In December 2013, symptoms suggestive of phytoplasma infection were noticed on flax in different experimental fields of Central Research Institute for Jute and Allied Fibres (CRIJAF) research farm, Barrackpore, India, and the incidence was less than 2%. Because incidence of phytoplasma diseases are increasing worldwide, occurrence of a phytoplasma in a new geographical area poses an imminent threat. The infected plants showed floral virescence, phyllody, and stem fasciation (flattened stem). Floral malformation was very conspicuous with abnormal structures replacing normal flowers. All the floral parts, including petals, turned into green leaves. Total DNA was extracted from leaf mid veins of three symptomatic and three asymptomatic plants using a DNeasy Plant Mini Kit (Qiagen). PCR was carried out with the phytoplasma-specific universal P1/P7 primer set followed by nested primer pair R16F2n/R16R2 (2), resulting in DNA amplicons that were 1.8 kb and 1.2 kb, respectively, in all symptomatic samples tested. No amplification was observed with DNA from symptomless samples. This suggested association of a phytoplasma with the disease. The five purified nested PCR products were cloned in a pGEM-T Easy vector (Promega) and sequenced. One of the sequences that proved to be identical to the others was deposited in GenBank (Accession No. KJ417660). The consensus sequence was analyzed by NCBI BLAST and found to share 99% similarity with the 16Sr DNA sequence of the 'Candidatus Phytoplasma asteris' reference strain (GenBank HQ828108), which belongs to 16SrI group. The phylogenetic tree based on 16SrDNA sequence of phytoplasmas belonging to group 16SrI and other distinct phytoplasma groups also showed that the phytoplasma clustered with members of group 16SrI (3). The nested PCR product of R16F2n/R16R2 was digested using restriction enzymes AluI, BfaI, BstU, HhaI, HpaI, KpnI, MseI, and RsaI. The RFLP patterns were compared with those of known phytoplasma strains (2) and they matched the patterns for aster yellows subgroup B (16Sr I-B). Subsequently, the iPhyClassifier 16Sr group/subgroup classification based on similarity (4) analyses showed that the studied strain had 16SrDNA sequences in the 16SrI-B group with a similarity coefficient of 1.00. To the best of our knowledge, this is the first report of 16SrI-B phytoplasma associated with flax in India.

Identification of potential leafhoppers vectors of phytoplasmas (16SrIII group) associated with broccoli stunt disease in Brazil

Recently, disease in broccoli plants (Brassica oleracea var. italica) was associated with three distinct phytoplasmas in Brazil. The disease named broccoli stunt (BS) has caused significant economic losses in Sao Paulo State. Group 16SrIII phytoplasmas is the most common group that have been associated with BS. The BS disease is still poorly understood, and the lack of information about the vectors further impairs its management. In this study, leafhoppers belonging to 18 different species were collected from weeds thriving near broccoli fields that were affected by group 16SrIII phytoplasmas. Specific primers revealed the presence of group 16SrIII phytoplasmas in five leafhoppers: Empoasca spp., Agallia albidula Uhler, Agalliana sticticollis (Stal), Planicephalus flavicosta (Stal), and Atanus nitidus (Linnavuori). The identity of the phytoplasmas was confirmed through DNA sequencing analysis. The leafhoppers were infected by phytoplasmas of the 16SrIII group (proposed “Candidatus Phytoplasma pruni” species) and are phylogenetically related to the broccoli stunt phytoplasma (BSP) strains detected in the study area and, thus, are considered potential vectors of group 16SrIII phytoplasmas to broccoli plants.

First Report of a 16SrV-C Phytoplasma Causing Little Leaf and Bunchy Top of Tossa Jute (Corchorus olitorius) in India.

Jute is the most important phloem fiber crop of the world, and is mainly grown in the South East Asian countries of India, Bangladesh, Nepal, China, Indonesia, Thailand, and Myanmar, and few South American countries. The fiber is used in making sacks, ropes, bags, carpets, shoes, geo-textiles, and home decorations. There are two kinds of jute: tossa jute (Corchorus olitorius L.) and white jute (C. capsularis). In June 2012, symptoms suggestive of phytoplasma infection (little leaf and bunchy top) were noticed on tossa jute in different experimental fields of the CRIJAF research farm, Barrackpore, India, and the incidence of the disease varied from 5 to 20%. The infected plants showed profuse lateral branching with a bushy appearance. In many plants, branching at the apical portion developed a bunchy top symptom with tufts of smaller leaves. Leafy stem was also common in many plants with main stems covered with numerous little leaves. Total DNA was extracted from leaf midveins of 15 symptomatic and 5 asymptomatic plants by using an improved salt concentration and simple sodium acetate CTAB method (1). PCR was carried out with universal P1/P7 primer set followed by nested primer pair R16F2n/R16R2 (3), resulting in DNA amplicons that were 1.8 kb and 1.2 kb, respectively, in all symptomatic samples tested. Phytoplasma was not detected in symptomless samples. The five purified nested products were cloned in a pGEM-T Easy vector (Promega) and sequenced. One of the sequences that proved to be identical was deposited in GenBank (Accession No. KF501045). The consensus sequence was analyzed by NCBI BLAST and found to share 99% similarity with the 16Sr DNA sequence of the alder yellows phytoplasma reference strain (GenBank Accession No. AY028789), which belongs to the 16SrV group. The phylogenetic tree based on the 16SrDNA sequence of phytoplasmas belonging to group 16SrV and other distinct phytoplasma groups also showed that the phytoplasma clustered with members of subgroup 16SrV (4). Subsequently, in silico RFLP analysis of the nested PCR product with the pDRAW32 program using AluI and TruI restriction site used for 16SrV subgroups A, B, C, D, and E indicated that the 16SrV Corchorus strain belonged to subgroup C. RFLP patterns from all symptomatic C. olitorius samples were identical to the 16SrV-C pattern (2). The vector species transmitting the concerned phytoplasma in C. olitorius still needs to be identified. The leaf hopper, Amrasca biguttula biguttula, may be a potential vector as it is often noticed in jute fields. To the best of our knowledge, this is the first report of 16SrV-C phytoplasma associated with tossa jute (C. olitorius) in India. Initiative has to be taken to manage this disease; otherwise, branching of the main stems would badly affect the fiber quality as well as yield.

Aster yellows group (16SrI), subgroups 16SrI‐A and 16SrI‐B, phytoplasmas associated with lettuce yellows in Texas

Lettuce (Lactuca sativa) is an important vegetable crop grown in the United States. Lettuce yellows (previously termed lettuce aster yellows) is a devastating disease commonly found in lettuce fields and causes significant economic loss to growers, damaging 40-100% of the crop in some seasons (Thompson, 4). The causal pathogen was not verified until a phytoplasmal aetiology was confirmed by molecular means (Lee et al., 1). In 2013, an epidemic of lettuce yellows occurred in the Winter Garden region of Texas. The infected plants were stunted with blanching and chlorosis in young heart leaves (Fig. 1). A total of thirteen samples from two different farms, including three apparently lacking symptoms from Romaine and leaf lettuce cultivars, were collected and tested for phytoplasmas. DNA was extracted from leaf veinal tissue, and nested PCRs using universal primer pair P1/16S-SR, followed by R16F2n/R16R2n and P1A/16S-SR, were performed as described previously (Lee et al., 2) to detect the presence of phytoplasmas in the samples. The taxonomic affiliation of the putative phytoplasmas detected was determined by RFLP analysis using nested PCR products (Lee et al., 1; Zhao et al., 5). A nested PCR using primer pair rpF1/rpR1 followed by rp(I)F1/rp(I)R1A as described previously (Lee et al., 2) was used to amplify a phytoplasma DNA segment (about 1.2 kb) of the ribosomal protein (rp) operon that encompassed genes rplV (rpl22) and rpsC (rps3); and a nested PCR using primer pair AYsecYF1/secYR1(I) followed by AYsecYF1/AYsecYR1 (Lee et al., 3) was used to amplify a phytoplasma DNA segment (about 1.3 kb) of the partial spc operon that includes the complete secY gene. Four PCR-amplified products from each of 16S rDNA, rp, and secY genes were cloned and sequenced as previously described (Lee et al., 2, 3), and deposited in GenBank (Accession Nos. KF573449-KF573456; rp, KF573441-KF573448; secY, KF573433-KF573440). The results indicated all 13 lettuce samples were infected with phytoplasmas. Both lettuce cultivars were infected with 16SrI-A and 16SrI-B phytoplasmas (Fig. 2). Phylogenetic analysis with 16S rRNA (Fig. 3), rp (Fig. 4) and secY gene (Fig. 5) sequences confirmed the presence of two distinct lineages (Fig. 3). This is the first report confirming that lettuce yellows in Texas is associated with two distinct phytoplasma strains. The finding implicates that multiple vectors may be involved in the disease outbreak. The information on identities of associated phytoplasmas should facilitate formulating effective strategies to combat this newly emerging lettuce yellows in Texas.