Phytoplasma Strains Research Articles

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.

A Multiplex-PCR Method for Diagnosis of AY-Group Phytoplasmas.

Polymerase chain reaction (PCR) methods using phytoplasma-specific primers are widely used to detect phytoplasmas from infected plants and insects. Here, I describe a method of multiplex-PCR to amplify nine gene fragments in PCR reactions from AY-group phytoplasmas. Strain-identification was possible after electrophoresis and direct sequencing was also possible after PCR. The combinations of primers can be easily modified, so this method could be applied to other phytoplasma strains.

16SrII phytoplasma associated with date palm and Mexican fan palm in Saudi Arabia

Date palm and Mexican fan palm trees showing symptoms previously associated with phytoplasmas were observed in the Al-Qassim region, Saudi Arabia in 2017. DNA amplification, sequencing, and phylogenetic analysis revealed the presence of a ‘Candidatus Phytoplasma australasia’- related strain in eighteen of the eighty-three symptomatic plants collected that were positive using 16S-based assays. This study confirmed the presence of phytoplasma affecting date palms in Saudi Arabia and it is the first report of several date palm cultivars associated with ‘Ca. P. australasia’-related strains. This is also the first report worldwide of Mexican fan palm trees affected by 16SrII phytoplasma strains and the first report of this plant host affected by phytoplasmas in Saudi Arabia. The implications of these findings are vital to implement management strategies and avoid economic losses in Saudi Arabia and the Middle East, which are the main producers of dates in the world.

Detection and characterisation of phytoplasma strains associated with field bindweed witches’ broom disease in Iran

During 2013–2015 surveys in Fars, Lorestan and Yazd provinces (Iran), a field bindweed witches’ broom (FBWB) disease was observed. The main symptoms were reduction of leaves size, yellowing, internode shortening, witches’ broom and stunting. The agent of FBWB was dodder transmitted to periwinkle plants inducing phytoplasma-type symptoms. Amplifications of nearly 1.8 and 1.2 kbp were, respectively, obtained from 15 symptomatic bindweed plants and 28 symptomatic dodder-inoculated periwinkles. Virtual RFLP analyses showed that the phytoplasma detected belonged to 16SrXII-A subgroup, and it was the same in all the samples examined; phylogenetic analyses confirmed it as a ‘Candidatus Phytoplasma solani’-related strain. This is the first report of 16SrXII-A phytoplasmas presence in bindweed plants showing witches’ broom symptoms in Fars, Lorestan and Yazd provinces. As a perennial widespread weed, it may act as a 16SrXII-A phytoplasma source for alfalfa, grapevine, Sophora alopecuroides, tomato, hemp and Japanese spindle reported diseases in these Iranian provinces.

Potato purple top disease associated with the novel subgroup 16SrII-X phytoplasma.

Potato (Solanum tuberosum) is a very economically important perennial tuberous crop in Saudi Arabia. Potato plants displaying symptoms associated with potato purple top disease, such as aerial tubers and purple and small leaves, were observed in Al-Bukairiyah, Fowlq and Buraydah, Al-Tarafiyah, Qassim governorate, Saudi Arabia. In this study, we examined samples taken from 12 symptomatic potato plants and confirmed the presence of phytoplasma DNA. Analysis of the 16S rRNA-encoding sequences revealed that the symptomatic plants were infected with phytoplasma belonging to the peanut witches'-broom group (16SrII). Sequencing of the 16S rRNA- encoding gene, computer-simulated RFLP analysis and phylogenetic analysis revealed the presence of a novel representative of the 16SrII-X subgroup. The present study identified potato plants as a novel host for novel phytoplasma strains belonging to the pigeon pea witches'-broom group in Saudi Arabia.

Apricot yellows associated with ‘ Candidatus Phytoplasma phoenicium’ in Iran

Almond witches’ broom associated with ‘ Candidatus Phytoplasma phoenicium’ is an economically important disease of almond in Iran and Lebanon. During surveys of almond witches’ broom in 2012–2015, an apricot yellows disease was observed in Fars Province of Iran. The characteristic symptoms of the disease were leaf yellowing, inward leaf curl, scorch of leaf margins, shortened internodes, production of rosettes at the tips of the branches, and decline, stunting, and death of affected trees. Healthy bitter almond and apricot seedlings, grafted with shoots from symptomatic trees, exhibited phytoplasma-type symptoms. A 16S rDNA fragment of 1,250 bp was amplified by nested-PCR from affected trees and grafted seedlings. Nucleotide sequence identity, presence of species-specific signature sequences, and phylogenetic analysis of 16S rDNA allowed the assignation of the phytoplasma strains identified to the ‘ Ca . P. phoenicium’. In vitro and in silico RFLP analyses of the amplified fragment allowed affiliation of the apricot yellows phytoplasma to a molecular variant in the subgroup 16SrIX-B. Within the population strains identified in this and previous studies, 16 genetic lineages were determined within 16S rDNA nucleotide sequences by the combination of 19 single nucleotide polymorphisms. The apricot yellows phytoplasma strains belong to a unique genetic lineage distinguished by the presence of three lineage-specific SNPs. This first report of ‘ Ca . P. phoenicium’ in association with apricot yellows in Iran opens new perspectives on the epidemiology of almond witches’ broom, suggesting possible adaptation of the phytoplasma to other fruit tree species.

Alterations of plant architecture and phase transition by the phytoplasma virulence factor SAP11.

As key mediators linking developmental processes with plant immunity, TCP (TEOSINTE-BRANCHED, CYCLOIDEA, PROLIFERATION FACTOR 1 and 2) transcription factors have been increasingly shown to be targets of pathogenic effectors. We report here that TB/CYC (TEOSINTE-BRANCHED/CYCLOIDEA)-TCPs are destabilized by phytoplasma SAP11 effectors, leading to the proliferation of axillary meristems. Although a high degree of sequence diversity was observed among putative SAP11 effectors identified from evolutionarily distinct clusters of phytoplasmas, these effectors acquired fundamental activity in destabilizing TB/CYC-TCPs. In addition, we demonstrate that miR156/SPLs and miR172/AP2 modules, which represent key regulatory hubs involved in plant phase transition, were modulated by Aster Yellows phytoplasma strain Witches' Broom (AY-WB) protein SAP11. A late-flowering phenotype with significant changes in the expression of flowering-related genes was observed in transgenic Arabidopsis plants expressing SAP11AYWB. These morphological and molecular alterations were correlated with the ability of SAP11 effectors to destabilize CIN (CINCINNATA)-TCPs. Although not all putative SAP11 effectors display broad-spectrum activities in modulating morphological and physiological changes in host plants, they serve as core virulence factors responsible for the witches' broom symptom caused by phytoplasmas.

Genotyping Points to Divergent Evolution of 'Candidatus Phytoplasma asteris' Strains Causing North American Grapevine Yellows and Strains Causing Aster Yellows.

Grapevine yellows diseases occur in cultivated grapevine (Vitis vinifera L.) on several continents, where the diseases are known by different names depending upon the identities of the causal phytoplasmas. In this study, phytoplasma strains associated with grapevine yellows disease (North American grapevine yellows [NAGY]) in vineyards of Pennsylvania were characterized as belonging to 16S ribosomal RNA (rRNA) gene restriction fragment length polymorphism group 16SrI (aster yellows phytoplasma group), subgroup 16SrI-B (I-B), and variant subgroup I-B*. The strains (NAGYI strains) were subjected to genotyping based on analyses of 16S rRNA and secY genes, and to in silico three-dimensional modeling of the SecY protein. Although the NAGYI strains are closely related to aster yellows (AY) phytoplasma strains and are classified like AY strains in subgroup I-B or in variant subgroup I-B*, the results from genotyping and protein modeling may signal ongoing evolutionary divergence of NAGYI strains from related strains in subgroup 16SrI-B.

Differential expression and phytohormone unbalance in Citrus aurantifolia plants during “sudden decline of lime”, a new phytoplasma disease of citrus

For decades, the production of acid lime (Citrus aurantifolia) in Oman has been affected by diseases caused by phytoplasmas, notably witches’ broom disease of lime (WBDL). In recent years, a new phytoplasma strain in Oman has been observed that promotes the sudden decline of lime (SDL). The molecular mechanisms behind its pathogenicity and mode of interaction with citrus host plants is still completely unknown. In this study, we evaluated the differential expression of genes in symptomatic and asymptomatic lime trees in Oman using a real-time quantitative PCR assay. Among 27 regulatory and biosynthesis-related genes tested in Citrus aurantifolia plants during phytoplasma infection, we verified the presence of 14 responsive genes in plants showing SDL symptoms, revealing a specific set of SDL-responsive genes. Quantification data of endogenous 3-indoleacetic acid and jasmonic acid show an unbalanced hormonal content in symptomatic lime trees, corroborating the gene expression data. The identification of regulatory genes differentially expressed in plant-phytoplasma interactions during SDL will help to elucidate the mechanisms possibly involved in defense responses, development and death-triggered signals in infected citrus plants.

Characterization of a 16SrII subgroup D phytoplasma strain associated with Calendula officinalis phyllody in Iran.

Calendula officinalis plants with phyllody symptoms (CaoP) were observed in Yazd and Ashkezar (Yazd province, Iran) during 2013-2016. Twenty-one symptomatic and four asymptomatic plants were transferred individually to the greenhouse and potted for the biological and molecular characterization of associated phytoplasma. The dodder transmission from symptomatic potted marigold plants, induced virescence, phyllody and witches' broom symptoms in periwinkle. Total DNAs extracted from symptomatic and symptomless plants and dodder-inoculated periwinkles were subjected to nested PCR assay using primer pairs amplifying phytoplasma ribosomal DNA. Expected PCR amplification was detected in all CaoP plant and dodder-inoculated periwinkle samples. RFLP analysis of the amplicons obtained in direct PCR with P1/P7 primers using RsaI, AluI, MseI, HinfI and HaeIII restriction enzymes showed profiles identical to each other and related to phytoplasmas in all the 21 positive samples. R16mF2/R16mR2 amplicons from six CaoP plant samples were sequenced where consensus sequences had 100% of identity among each other. R16F2n/R16R2-trimmed sequences (1248bp) of representative samples from Yazd and Ashkezar were deposited in GenBank under accession numbers KU297202 and MH065715, respectively. BLAST search and phylogenetic analysis showed that the CaoP phytoplasma had 99% homology and clusters with phytoplasmas in group 16SrII. Computer-simulated analysis using iPhyClassifier suggests that the CaoP RFLP 16S rRNA gene pattern was identical to 16SrII-D phytoplasmas subgroup. Phytoplasma strains (16SrII-D) were reported as alfalfa witches' broom disease agentpreviously in the same geographic areas, and it is possible that alfalfa plays a role in the epidemiology of CaoP disease or vice-versa.

A ‘Candidatus Phytoplasma omanense’-related strain detected in yellowing grapevine, stunted bindweed and Cixiidae planthoppers in Lebanon

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.

Increasing incidence of tomato big bud phytoplasma in Ranga Reddy District of Telangana State, India

Tomato is an important vegetable crop and India ranks second in area and production of tomato worldwide. During field surveys in Moinabad and Shamshabad Mandals in Ranga Reddy district of Telangana from 2014–2016, symptoms like big bud and phyllody were observed on tomato. The affected plants did not produce any fruit. The phytoplasma strains were detected and characterized using universal and nested primer pairs of phytoplasma with amplification of 16S rRNA and secA genes. BLAST analysis of 1.25 kb 16S rDNA partial sequences of nested PCR products and 880 bp of secA gene products obtained from symptomatic TBB (tomato big bud) samples revealed 99% sequence identity with strains of ‘Ca. Phytoplasma australasia’ (16Sr II group). Phylogenetic analysis and virtual RFLP analysis of 16SrDNA sequences of tomato big bud phytoplasma (TBBP) strain also suggested the closest relationship with ‘Ca. P. australasia’16Sr II-D subgroup related strain. Present study confirmed association of 16Sr II-D subgroup of phytoplasma associated with tomato big bud disease in Telangana State of India.

Genetic analysis of 'Candidatus Phytoplasma aurantifolia' associated with witches' broom on acid lime trees.

“Candidatus Phytoplasma aurantifolia” is associated with witches’ broom disease of lime in Oman and the UAE. A previous study showed that an infection by phytoplasma may not necessarily result in the physical appearance of witches’ broom symptoms in some locations in Oman and the UAE. This study investigated whether phytoplasma strains belonging to “Ca. P. aurantifolia” (based on the 16S rRNA gene analysis) in locations where disease symptoms are expressed are different from phytoplasma in locations where disease symptoms are not expressed. About 21 phytoplasma strains (15 from areas and trees with disease symptoms and six from areas and trees without disease symptoms) were included in the analysis. The study utilized sequences of the imp and SAP11 genes to characterize the 21 strains. Phylogenetic analysis of both genes showed that the 21 strains are similar to each other and to reference strains in GenBank. The study shows that there is a low level of diversity among all phytoplasma strains. In addition, it shows that phytoplasma in places where witches’ broom symptoms are not expressed are similar to phytoplasma in places where disease symptoms are expressed. This may suggest that disease expression is not linked to the presence of different phytoplasma strains, but may be due to other factors such as weather conditions.

Identification and characterization of Candidatus Phytoplasma trifolii (16SrVI-D) inducing shoot proliferation disease of potato in India

Potato (Solanum tuberosum) is a widely cultivated and commercially important vegetable crop widely grown in India. Profuse shoot proliferation symptoms were observed in potato plants of two varieties, Kufri Khyati and Kufri Surya at the Research plots of All India Co-ordinated Research Project on Potato, Orissa University of Agriculture and Technology, Bhubaneswar, Odisha, India during February, 2017. The symptomatic plants were tested for phytoplasma etiology. Nested and semi-nested PCR assays primed by primer pairs P1/P7 and 3Far/3Rev for 16S rRNA gene and secAF1/R3 for secA gene amplified DNA fragments of 1.3 kb (3Far/3Rev) and 840 bp (secAF1/R3), respectively from all the symptomatic potato samples but not from any asymptomatic plants. The 16S rDNA sequences of potato tuber (Acc. no. KY815100, Kufri Khyati; MG566064, Kufri Surya) and shoot phytoplasma strains (Acc. no. KY815099, Kufri Khyati; MG566063, Kufri Surya) shared 100% nucleotide sequence identity with 16S rDNA sequence of identified phytoplasma strains belonging to 16SrVI-D subgroup associated with Lens culinaris witches’ broom (Acc. no. KY439869), brinjal little leaf (Acc. no. KX284698) and Brassica oleracea var. capitata witches’ broom (Acc. no. KX671553) in pair wise sequence comparison. Sequence comparison of 840 bp of secA gene of potato phytoplasma strains from tuber of variety, Kufri Khyati (Acc. no. KY815101) and variety Kufri Surya (Acc. no. MG566065) showed 99% sequence identity to secA gene sequences of brinjal little leaf phytoplasma strains (Acc. no. KX610808, KX894792, KY064175), which belonged to 16SrVI-D subgroup. Phylogenetic analysis of 16S rRNA and secA gene sequences, and virtual RFLP analysis of 16S rDNA sequences of potato shoot proliferation phytoplasma strain confirmed their clustering and grouping with strains of clover proliferation subgroup D. Our results confirmed potato as a new host of 16SrVI-D subgroup of phytoplasmas in the world. The 16SrVI-D strain is wide spread in India and cause significant yield losses and hence poses serious socio-economic threat to commercially grown potato crops.

Identification of ‘Candidatus Phytoplasma asteris’ causing sesame phyllody disease and its natural weed host in Jammu, India

During May–June 2016, little leaf and phyllody symptoms were observed on 8% of sesame crop in Chakbhalwal and Kotbhalwal regions of Jammu, India. Phytoplasma suspected witches’ broom symptoms on Cannabis sativa subsp. sativa was also observed in and around sesame fields with the incidence varied from 5 to 12% in different fields. Total genomic DNA from both symptomatic sesame and Cannabis plants were subjected to nested PCR assays using two sets of universal phytoplasma specific primers P1/P7 followed by R16F2n/R16R2 and 3Far/3Rev to amplify the 16S rDNA fragments. First round PCR amplification with primer pair P1/P7 did not yield expected 1.8 kb products of 16S rRNA gene region from any of the symptomatic test samples. However, DNA specific fragments of approximately 1.2 and 1.3 kb were amplified from sesame and C. sativa sub sp. sativa by using R16F2n/R16R2 and 3Far/3Rev primer pairs, respectively. BLASTn comparison and phylogenetic analysis of 16SrDNA phytoplasma sequences of sesame and cannabis phytoplasma strains revealed the association of ‘C. Phytoplasma asteris’ (16Sr I group) with symptomatic samples. The virtual RFLP analysis of the 16Sr DNA sequence of sesame and cannabis further identified as phytoplasma strains of 16SrI-B subgroup.

Molecular detection and characterization of a stolbur phytoplasma associated with Narcissus tazetta phyllody in Iran

AbstractDuring field surveys in 2015, a phytoplasma‐associated disease was identified in Narcissus tazetta plants in Behbahan, Iran. The characteristic symptoms were phyllody and virescence. The presence of phytoplasma in symptomatic plants was confirmed using PCR amplification and sequencing of 16S rRNA, tuf, secY and vmp1 genes. Based on the blastn results, the sequences of 16S rRNA, tuf, secY and vmp1 genes shared, respectively, 99%, 100%, 99% and 99% sequence identity with phytoplasma strains in 16SrXII‐A subgroup. RFLP and phylogenetic analyses using the sequences of 16S rRNA, tuf and secY genes confirmed the assortment of studied strains to 16SrXII‐A phytoplasma subgroup. Sequence comparison of these four genes revealed that all the sequences of 28 strains studied were identical. To the best of our knowledge, the association of “Candidatus Phytoplasma solani” with N. tazetta was demonstrated for the first time in the world.

Molecular characterization of phytoplasmaassociated with four important ornamental plant species in India and identification of natural potential spread sources.

Phytoplasma suspected symptoms of phyllody, witches' broom, leaf yellowing, stunting and little leaf were observed in Chrysanthemum morifolium, Bougainvillea glabra, Jasminum sambac and Callistephus chinensis during survey of flower nurseries and experimental ornamental fields at Delhi, Maharashtra, Tamil Nadu and Karnataka from 2014 to 2016. Pleomorphic bodies typical to phytoplasma structures were observed in the phloem sieve elements of ultrathin sections of all the four symptomatic ornamental plants (stem tissue) in transmission electron microscope. Amplification of 1.8 and 1.2kb phytoplasma DNA products was observed in all the four test plants in PCR assays using universal primer pairs P1/P7 followed by nested primer pair R16F2n/R16R2, respectively. Pairwise sequence comparison, phylogeny and virtual RFLP analysis of 16S rDNA sequences confirmed the association of two phytoplasma subgroups (16SrI-B and 16SrII-D) in four ornamental plant species. 'Ca. P. aurantifolia' subgroup D (16SrII-D) was found associated with chrysanthemum phyllody and leaf yellowing at Delhi and Tamil Nadu, bougainvillea little leaf and yellowing at Delhi and Chinese aster phyllody at Bengaluru, Karnataka. However, jasmine little leaf and yellowing at Bengaluru, Karnataka and chrysanthemum stunting at Pune were found to be associated with 'Ca. P. asteris' subgroup B-related strains (16SrI-B). The identification of 16SrII-D subgroup phytoplasma infecting bougainvillea and 16SrI-B subgroup infecting jasmine are the new reports to the world. Besides weed species, Cannabis sativa showing witches' broom in jasmine fields at Bengaluru and Parthenium hysterophorus showing witches' broom symptoms in chrysanthemum fields at Delhi were identified to be caused by phytoplasma strains classified under subgroups 16SrI-B and 16SrII-D, respectively, by PCR assays and 16Sr DNA sequence comparison analysis. Among the three major leafhopper species identified, only Hishimonas phycitis was identified positive for 16SrI-B and 16SrII-D subgroups of phytoplasmas from chrysanthemum fields at Delhi and jasmine fields at Bengaluru, respectively. The identity of similar phytoplasma strains infecting ornamental species in leafhopper and the weed species in the present study suggested that H. phycitis and weeds may act as potential natural sources for secondary spread of the identified phytoplasma strains.

Molecular characterization of 'Clover proliferation' phytoplasma subgroup-D (16SrVI-D) associated with vegetables crops in India.

Nine vegetable plants species exhibiting phytoplasma suspected symptoms of white/purple leaf, little leaf, flat stem, witches' broom, phyllody and leaf yellowing were observed in experimental fields at Indian Agricultural Research Institute, New Delhi from December 2015 to July 2016. Total DNA extracted from the three healthy and three symptomatic leaves of all the nine vegetables were subjected to PCR assays using phytoplasma specific primers P1/P7 followed by R16F2n/R16R2 and 3Far/3Rev to amplify the 16S rDNA fragments. No amplifications of DNA were observed in first round PCR assays with primer pair P1/P7 from any of the symptomatic samples. However, phytoplasma DNA specific fragments of~1.3kb were amplified from Apium graveolens L. (two isolates), Brassica oleracea vr. capitata L. (one isolate) and Solanum melongena L. (one isolate) by using 3Far/3Rev primer pair and 1.2kb fragment was amplified from Lactuca sativa L. (one isolate) by using R16F2n/R16R2 primer pair. No DNA amplification was seen in other symptomatic vegetable samples of tomato, carrot, cucurbit,bitter gourd and Amaranthus species utilizing either P1/P7 primer pair followed by 3Far/3Rev or R16F2n/R16R2 primer pairs. Out of three leafhopper species collected from the symptomatic vegetable fields, only Hishimonus phycitis was found positive for association of phytoplasma. No DNA amplifications were observed in healthy plant samples and insects collected from non-symptomatic fields. Comparative sequence comparison analyses of 16S rDNA of positive found vegetable phytoplasma strains revealed 100% sequence identities among each other and with phytoplasma strains of 'clover proliferation' (16SrVI) group. Phytoplasma sequences, virtual RFLPs and phylogenetic analyses of 16S rDNA sequence comparison confirmed the identification of 16SrVI subgroup D strain of phytoplasmas in four vegetablesand one leafhopper (HP) species. Further virtual RFLP analysis of 16S rDNA sequence of the vegetables phytoplasma strains confirmed their taxonomic classification with strains of 'clover proliferation' subgroup D. Since, H. phycitis feeding on symptomatic vegetable species in the study was also tested positive for the 16SrVI phytoplasma subgroup-D as of vegetables; it may act as potent natural reservoir of16SrVI-D subgroup of phytoplasmas infecting vegetable and other important agricultural crops.

Molecular identification and characterization of the new 16SrIX-J and cpn60 UT IX-J phytoplasma subgroup associated with chicory bushy stunt disease in Saudi Arabia.

Chicory (Cichorium intybus) is a perennial plant (Asteraceae) that grows wild in pasture fields in Saudi Arabia. Chicory plants displaying symptoms typically induced by phytoplasmas, such as bushy phenotype and stunt, were observed in the Mulayda region, Qassim governorate, Saudi Arabia. In this study we examined samples taken from three symptomatic chicory plants and confirmed the presence of phytoplasma DNA. Analysis of the 16S rRNA-encoding sequences showed that the plants were infected with a phytoplasma from the pigeon pea witches'-broom group (16SrIX). Sequencing of the 16S rRNA-encoding gene and the partial cpn60 sequence, computer-simulated RFLP analysis, and phylogenetic analysis of both markers revealed that the phytoplasma identified was representative of a new 16SrIX-J and cpn60 UT IX-IJ subgroup. The present study identified chicory plants as a novel host for phytoplasma strains within the pigeon pea witches'-broom phytoplasma group, and expanded the known diversity of this group.

Presence of 16SrIV phytoplasmas of subgroups A, D and E in planthopper Haplaxius crudus Van Duzee insects in Yucatán, Mexico.

The present study was carried out to determine if group 16SrIV phytoplasmas, causing lethal yellowing (LY) disease, are present in Haplaxius crudus Van Duzee (Hemiptera: Cixiidae) insects associated with palms in Yucatán, Mexico. Haplaxius crudus feral insects were captured from palm foliage at two locations (Chicxulub Puerto and CICY, Mérida, where LY-type diseases are active) and evaluated individually for the presence of phytoplasma DNA by a group 16SrIV-specific nested PCR assay. The results showed positive detection in H. crudus insects in a proportion of 2.7% (of the total 2726 analyzed) during a 3-year period of study. The percentage of detection was different for each site, 5.9% positive of 799 insects from Mérida and 1.7% of 1927 from Chicxulub Puerto. Positive detections were also obtained in extracts from 5.3 to 1.2% of males and females, respectively. Sequencing and in silico RFLP and phylogenetic analyses of PCR-amplified rDNA products indicated that H. crudus insects from Chicxulub Puerto harbored phytoplasma strains of subgroups 16SrIV-A or 16SrIV-D, whereas in insects from Mérida the strains found were 16SrIV-A, 16SrIV-D or 16SrIV-E. The diversity of subgroup strains detected in H. crudus coincided with strains previously identified in palms showing LY-type disease syndromes in Yucatán thereby implicating H. crudus as a candidate vector of 16SrIV phytoplasmas in this region of Mexico.