Molecular Characterization of a Phytoplasma Associated with Potato Witches’‐broom Disease in Iran

Potato plants showing symptoms suggestive of potato witches’‐broom disease including witches’‐broom, little leaf, stunting, yellowing and swollen shoots formation in tubers were observed in the central Iran. For phytoplasma detection, Polymerase Chain Reaction (PCR) and nested PCR assays were performed using phytoplasma universal primer pair P1/P7, followed by primer pair R16F2n/R16R2. Random fragment length polymorphism analysis of potato phytoplasma isolates collected from different production areas using the CfoI restriction enzyme indicated that potato witches’‐broom phytoplasma isolate (PoWB) is genetically different from phytoplasmas associated with potato purple top disease in Iran. Sequence analysis of the partial 16S rRNA gene amplified by nested PCR indicated that ‘Candidatus Phytoplasma trifolii’ is associated with potato witches’‐broom disease in Iran. This is the first report of potato witches’‐broom disease in Iran.     

Molecular Characterization of Phytoplasma Associated with Rose Witches'-Broom in China

In 2005, rose plants (Rosa rugosa cv. ‘Plena’) exhibiting typical phytoplasma disease symptoms of stunting, yellowing, witches’‐broom and dieback were observed in Pingyin, Shandong Province, China. The disease, rose witches’‐broom (RoWB), is progressively destructive and can be graft‐transmitted. Polymerase chain reaction (PCR), sequencing of PCR products and electron microscopy were used to investigate the possible association of phytoplasma with RoWB. All results indicated that presence of phytoplasma in the symptomatic rose plants. Sequence alignment of 16S rRNA gene, tuf gene and rp gene confirmed that the phytoplasma associated with RoWB is the causal agent of Paulownia witches’‐broom disease, which might be transmitted from the paulownia tree that is several meters away. To our knowledge, this is the first report of the molecular characterization of phytoplasma infecting rose in China.     

Witches'Broom of Sarothamnus scoparius: A New Disease Associated with a Phytoplasma Related to the Spartium Witches'Broom Agent

In southern and central Italy, a witches’broom and decline disease of Sarothamnus scoparius has been observed. In affected plants, phytoplasmas were detected by PCR amplification of ribosomal DNA. Restriction fragment length polymorphism analysis of PCR-amplified DNA revealed that the diseased plants were infected by a phytoplasma that is closely related to the spartium witches’broom phytoplasma, a member of the apple proliferation group.     

Detection and identification of phytoplasma associated with witches’ broom and little leaf disease in Arundo donax: first report from India

During the survey of two successive years 2012–2013, in nearby places of Gorakhpur districts, Uttar Pradesh, India, Arundo donax plants were found to be exhibiting witches’ broom, excessive branching accompanied with little leaf symptoms with considerable disease incidence. Nested PCR carried out with universal primers pair R16F2n/R16R2 employing the PCR (P1/P7) product as a template DNA (1:20) resulted in expected size positive amplification ~1.2 kb in all symptom-bearing plants suggested the association of phytoplasma with witches’ broom disease of Narkat plants. BLASTn analysis of the 16S rRNA gene sequence showed the highest (99%) sequence identity with Candidatus phytoplasma asteris (16SrI group). In phylogenetic analysis, the sequence data showed close relationships with the members of 16SrI phytoplasma and clustered within a single clade of 16SrI group and closed to B subgroup representatives. This is a first report of 16Sr I-B group phytoplasma associated with witches’ broom accompanied with little leaf disease of Narkat in India.     

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

Abstract

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.     

Detection, Identification and Molecular Characterization of a Phytoplasma Associated with Arabian Jasmine (Jasminum sambac L.) Witches' Broom in Oman

Arabian jasmine (Jasminum sambac L.) plants showing witches’ broom (WB) symptoms were found in two regions in the Sultanate of Oman. Polymerase chain reaction (PCR) amplification of the 16S rRNA gene and the 16S–23S spacer region utilizing phytoplasma‐specific universal and designed primer pairs, and transmission electron microscopy of phytoplasma‐like structures in phloem elements confirmed phytoplasma infection in the symptomatic plants. PCR products primed with the P1/P7 primer pair were 1804 bp for jasmine witches’ broom (JasWB) and 1805 bp for alfalfa (Medicago sativa L.) witches’ broom (AlfWB). Actual and putative restriction fragment length polymorphic analysis indicated that jasmine and AlfWB phytoplasmas were molecularly indistinguishable from each other and closely related to papaya yellow crinkle (PYC), as well as being distinct from lime WB (LWB) and Omani alfalfa WB (OmAlfWB) phytoplasmas. A sequence homology search of JasWB and AlfWB showed 99.8% similarity with PYC from New Zealand and 99.6% similarity with each other (JasWB/AlfWB). The jasmine and AlfWB phytoplasmas were also shown to be related to the peanut WB group (16SrII) of 16S rRNA groups based on a phylogenetic tree generated from phytoplasma strains primed with the P1/P7 primer pair and representing the 15 phytoplasma groups.     

Mixed Infection and Natural Spread of ‘Candidatus Phytoplasma asteris’ and Mungbean Yellow Mosaic India Virus Affecting Soya Bean Crop in India

Suspected phytoplasma and virus‐like symptoms of little leaf, yellow mosaic and witches’ broom were recorded on soya bean and two weed species (Digitaria sanguinalis and Parthenium hysterophorus), at experimental fields of Indian Agricultural Research Institute, New Delhi, India, in August–September 2013. The phytoplasma aetiology was confirmed in symptomatic soya bean and both the weed species by direct and nested PCR assays with phytoplasma‐specific universal primer pairs (P1/P6 and R16F2n/R16R2n). One major leafhopper species viz. Empoasca motti Pruthi feeding on symptomatic soya bean plants was also found phytoplasma positive in nested PCR assays. Sequencing BLASTn search analysis and phylogenetic analysis revealed that 16Sr DNA sequences of phytoplasma isolates of soya bean, weeds and leafhoppers had 99% sequence identity among themselves and were related to strains of ‘Candidatus Phytoplasma asteris’. PCR assays with Mungbean yellow mosaic India virus (MYMIV) coat‐protein‐specific primers yielded an amplicon of approximately 770 bp both from symptomatic soya bean and from whiteflies (Bemisia tabaci) feeding on soya bean, confirmed the presence of MYMIV in soya bean and whitefly. Hence, this study suggested the mixed infection of MYMIV and ‘Ca. P. asteris’ with soya bean yellow leaf and witches’ broom syndrome. The two weed species (D. sanguinalis and P. hysterophorus) were recorded as putative alternative hosts for ‘Ca. P. asteris’ soya bean Indian strain. However, the leafhopper E. motti was recorded as putative vector for the identified soya bean phytoplasma isolate, and the whitefly (B. tabaci) was identified as vector of MYMIV which belonged to Asia‐II‐1 genotype.     

Molecular Characterization and Potential Insect Vector of a Phytoplasma Associated with Garden Beet Witches' Broom in Yazd, Iran

In 2002, garden beet witches’ broom (GBWB) phytoplasma was detected for the first time in garden beet plants (Beta vulgaris L. ssp. esculenta) in Yazd, Iran. Nested polymerase chain reaction (PCR) and restriction fragment length polymorphic (RFLP) analysis of PCR‐amplified phytoplasma 16S rDNA were employed for the detection and identification of the phytoplasma associated with garden beet. A phytoplasma belonging to subgroup 16SrII‐E, in the peanut witches’ broom group (16SrII), was detected in infected plants. Asymptomatic plant samples and the negative control yielded no amplification. The result of analysis of the nucleotide sequence of a 1428 bp fragment of 16S rDNA gene from GBWB phytoplasma (GenBank accession number DQ302722 ) was basically consistent with the classification based on RFLP analysis, in which GBWB phytoplasma clustered with phytoplasmas of the 16SrII‐E subgroup. A search for a natural phytoplasma vector was conducted in Yazd in 2004, in an area where garden beet crops had been affected since 2002. The associated phytoplasma was detected in one leafhopper species, Orosius albicinctus, commonly present in this region. The leafhopper O. albicinctus was used in transmission tests to determine its vector status for the phytoplasma associated with GBWB. Two of eight plants that had been fed on by O. albicinctus, showed mild symptoms of GBWB including stunting and reddening of midveins. A phytoplasma was detected in the two symptomatic test plants by PCR using universal primers and it was identified by RFLP as the GBWB phytoplasma. This finding suggests O. albicinctus is a vector of the GBWB phytoplasma.     

Detection and Identification of Elm Yellows Group Phytoplasma (16SrV) Associated with Alfalfa Witches’ Broom Disease

In July, 2011, alfalfa plants were observed in Yangling, Shaanxi Province, China with typical witches’ broom symptoms. The presence of phytoplasma was confirmed by transmission electron microscopy and a nested PCR, which amplified a 1.2‐kb fragment using universal primer pairs P1/P6 followed by R16F2n/R2. Sequence, phylogeny and RFLP analyses showed that the alfalfa witches’ broom disease was associated with a phytoplasma of group 16SrV, subgroup V‐B. This is the first record of the 16SrV phytoplasma group infecting alfalfa plants.     

Molecular detection of Candidatus Phytoplasma spp. causing witches’ broom disease of acid lime (Citrus aurantifolia) in India

Phytoplasma infected acid lime plants in India develop characteristic symptoms like small chlorotic leaves, multiple sprouting and shortened internodes. Leaves drop prematurely and infected branches have distorted twigs resembling witches’ broom appearance which eventually show die-back symptoms. During its first report in 1999, witches’ broom disease identification was made on the basis of symptomatology and electron microscopy. However, molecular techniques have proved to be more accurate and reliable for phytoplasma detection than the conventional methods. During survey in the year 2010 six samples were collected from infected acid lime plants showing typical field symptoms from Vidarbha region of Maharastra. Initially, phytoplasma bodies were observed in phloem tissues of all six symptomatic samples under JEM 100S transmission electron microscope and all these six samples were subsequently screened using different set of phytoplasma specific universal primers by nested PCR, a widely recommended molecular technique for phytoplasma detection. In the present study P1/P7 “universal” phytoplasma-primer set was used for first round of PCR and amplified products were processed separately for nested PCR with three different nested primer pairs viz. R16F2n/R16R2, R16mF2/R16mR1 and fU5/rU3. The presence of phytoplasma was confirmed in all six suspected samples and one representative ~1.2 kb size amplicon was sequenced and deposited in GenBank as Candidatus Phytoplasma species AL-M (JQ808143). This is the first report of PCR based molecular detection of phytoplasma-induced witches’ broom disease of acid lime (WBDL) in India. Further molecular evaluation to determine the identity to the species level is in progress.     

Molecular Characterization of 16S Ribosomal DNA and Phylogenetic Analysis of Two X-disease Group Phytoplasmas Affecting China-tree (Melia azedarach L.) and Garlic (Allium sativum L.) in Argentina

Two phytoplasmas closely related to the X‐disease group were associated with China‐tree (Melia azedarach L.) and garlic (Allium sativum L.) decline diseases in Argentina. The present work was aimed at studying their phylogenetic relationship based on molecular characterization of the 16S ribosomal DNA sequences. Phytoplasma DNAs were obtained from naturally infected China‐tree and garlic plants from different geographical isolates. The results from analysis of restriction fragment length polymorphisms and nucleotide sequences of the 16S rDNA showed the affiliation of China‐tree and garlic decline phytoplasmas to the 16SrIII (X‐disease group), subgroups B and J, respectively. Both organisms had high sequence similarities in the 16SrDNA nucleotide sequence with the Chayote witches’ broom phytoplasma from Brazil. The phylogenetic tree, constructed by parsimony analysis, grouped the Garlic decline, China‐tree decline, Chayote witches’ broom and Clover yellow edge phytoplasmas into a cluster separated from the other phytoplasmas of the X‐disease group.     

Association of Elm Yellows Subgroup 16SrV‐B Phytoplasma with a Disease of Hovenia dulcis

Japanese raisin (Hovenia dulcis) trees with typical phytoplasma‐like symptoms were observed for the first time in South Korea. The disease, named Japanese raisin witches’ broom, is progressively destructive. The cause of the graft‐transmissible disease was confirmed by electron microscopy and molecular studies. The 16S rDNA sequence analysis showed that the phytoplasma was closely related to the elm yellows (EY) group, ribosomal subgroup 16SrV‐B. The 16S‐23S rDNA intergenic spacer region, fragment of rp operon and secY gene sequences had 96–99% similarity with members of EY phytoplasma. Based on the sequence analyses and phylogenetic studies, it was confirmed that the phytoplasma infecting Japanese raisin trees in Korea belongs to the EY group.     

Occurrence and Characterization of a 16SrII‐D Subgroup Phytoplasma Associated with Parsley Witches’ Broom Disease in Iran

During 2010–2013 surveys for the presence of phytoplasma diseases in Yazd province (Iran), a parsley witches’ broom (PrWB) disease was observed. Characteristic symptoms were excessive development of short spindly shoots from crown buds, little leaf, yellowing, witches’ broom, stunting, flower virescence and phyllody. The disease causative agent was dodder transmitted from symptomatic parsley to periwinkle and from periwinkle to periwinkle by grafting inducing phytoplasma‐type symptoms. Expected length DNA fragments of nearly 1800 and 1250 bp were, respectively, amplified from naturally infected parsley and experimentally inoculated periwinkle plants in direct polymerase chain reaction (PCR) using phytoplasma primer pair P1/P7 or nested PCR using the same primer pair followed by R16F2n/R16R2 primers. Restriction fragment length polymorphism and phylogenetic analyses of 16S rRNA gene sequences showed that the phytoplasma associated with PrWB disease in Yazd province belong to 16SrII‐D phytoplasma subgroup. This is the first report of association of a 16SrII‐related phytoplasma with PrWB disease in Iran.     

Changes in the composition of volatile compounds of Spartium junceum induced by the phytoplasmal disease,Spartium witches’‐broom

Abstract

In southern Italy, Spartium junceum (Spanish broom) is severely affected by a phytoplasmal disease, Spartium witches’‐broom (SpaWB). The volatile fractions extracted from flowers of healthy and diseased plants, examined by gas chromatography and gas chromatography–mass spectrometry, appeared to be quantitatively and qualitatively different. In both the healthy and the diseased plants, the main components were n‐alkanes, which occurred at a rate of 55.2% and 38.8%, respectively. The level of aliphatic acids was considerably lower in flowers of the diseased plants than in those of the healthy plants (4.5% vs. 18.7%). Sesquiterpenes were detected only in the diseased plants. It is possible that the changes in the composition of secondary metabolites of diseased plants can be related to plant defense responses.

Abbreviations: AP, apple proliferation; EY, elm yellows; SpaWB, Spartium witches’‐broom     

Characterization of 16SrII-D subgroup associated phytoplasmas in new host plants in Egypt

Samples of three plant species displaying phytoplasma symptoms were collected from Kafrelsheikh and Al-Gharbia governorates during 2014. Witches’ broom and virescence symptoms were observed in periwinkle (Catharanthus roseus). Onion (Allium cepa) plants showed yellowing, streaks and twisting and Opuntia abjecta with proliferation and cylindrical of cladodes. Total DNA was extracted from symptomatic and asymptomatic plants, and phytoplasma were detected in all 12 symptomatic plants collected through direct and nested PCR assays with primers P1/P7 and R16F2n/R16R2. The results of phylogenetic analysis revealed that the phytoplasma isolates belong to 16SrII group. With a nucleotide identity greater than 98.7% with three members of 16SrII group, Papaya yellow crinkle, Y10097; “Ca. P. aurantifolia”, U15442; and peanut witches’ broom, Al33765, the strains identified in this study are “Ca. P. aurantifolia”-related strains. Virtual RFLP analysis of the 16S rRNA gene sequences with 17 restriction enzymes confirmed that the phytoplasma isolates belong to the “Candidatus Phytoplasma australasia” 16SrII-D subgroup. To the best of our knowledge, periwinkle, onion and Opuntia abjecta are considered new hosts for 16SrII group in Egypt.     

Identification of phytoplasmas from Neoaliturus haematoceps associated with sesame phyllody disease in southwestern Turkey

Phytoplasmas were detected based on nested PCR of the F2nR2 region of the 16S rDNA from Neoaliturus haematoceps (Mulsant and Rey) (Family: Cicadellidae). A total of 65 insect samples collected from sesame fields in Antalya, Turkey, during 2012–2014 were tested for phytoplasma detection. Phytoplasmas detected in fifteen samples showed an amplicon approximately 1250 bp in size using the universal primers of P1/P7 and R16F2n/R16R2. Identification of the phytoplasmas by sequence analysis revealed three different 16S rDNA phytoplasma groups: the peanut witches’‐broom, group II; clover proliferation, group VI; and pigeon pea witches’‐broom, group IX. The molecular characterization of subgroups was determined by sequence analysis and PCR‐RFLP using the restriction enzymes RsaI and TaqI. Restriction profiles of the subgroups were also confirmed using the iPhyclassifier program. BLAST and PCR‐RFLP analyses classified the subgroups as II‐D, VI‐A and IX‐C. This is the first report of molecular detection of three 16S rDNA subgroups of phytoplasmas, II‐D, VI‐A and IX‐C, from N. haematoceps in Turkey. This study also supports earlier studies of sesame phyllody phytoplasmas by N. haematoceps.     

Molecular Identification of a Candidatus Phytoplasma asteris Associated with Cabbage Witches’‐Broom in China

In 2010, cabbages (Brassica oleracea L.) showing symptoms of proliferated axillary buds, crinkled leaves and plant stunting with shortened internodes typical to phytoplasma infection were found in a breeding facility in Beijing, China. Three symptomatic plants and one symptomless plant were collected, and total DNA was extracted from the midrib tissue and the flowers. With phytoplasma universal primers R16F2n/R16R2, a special fragment of 1247 bp (16S rDNA) was obtained from all three symptomatic cabbage plants, but not from the one symptomless cabbage plant. The 16S rDNA sequence showed 99% similarity with the homologous genes of the aster yellows group phytoplasma (16SrI group), and the phytoplasma was designed as CWBp‐BJ. Phylogenetic and computer‐simulated restriction fragment length polymorphism (RFLP) analysis of the 16S rDNA gene revealed that CWBp‐BJ belongs to subgroup 16SrI‐B. This is the first report of a phytoplasma associated with cabbage witches’‐broom in China.     

Association of a Phytoplasma with Pistachio Witches’ Broom Disease in Iran

Pistachio is an important crop in Iran, which is a major producer and exporter of pistachio nuts. The occurrence of a new disease of pistachio trees, characterized by the development of severe witches’ broom, stunted growth and leaf rosetting, was observed in Ghazvin Province. A phytoplasma was detected in infected trees by polymerase chain reaction (PCR) amplification of rRNA operon sequences. Nested PCR with primer pairs P1/P7 and R16F2n/R16R2 was used for specific detection of the phytoplasma in infected trees. To determine its taxonomy, the random fragment length polymorphism (RFLP) pattern and sequence analysis of the amplified rRNA gene were studied. Sequencing of the amplified products of the phytoplasma 16S rRNA gene indicated that pistachio witches’ broom (PWB) phytoplasma is in a separate 16S rRNA group of phytoplasmas (with sequence homology 97% in Blast search). The unique properties of the DNA of the PWB phytoplasma indicate that it is a representative of a new taxon.     

Occurrence,identification and transmission of the phytoplasma associated with tomato big bud disease and identification of its vector and weed host in Pakistan

Abstract

Tomato (Solanum lycopersicum L.) plants showing stunting, big bud, leaves yellowing or reddening and witches’-broom symptoms were observed since 2009 in Pakistan. A weed Parthenium hysterophorus grown in and around tomato fields also exhibited witches’-broom like symptoms. Fluorescence light microscopy of hand-cut stem stalk sections treated with Dienes’ stain showed blue areas in the phloem region of both tomato and P. hysterophorus symptomatic plants that indicated the association of phytoplasma with the complex. Amplification of 1.2?kb 16S rDNA fragment in nested PCR confirmed that the symptomatic tomato and P. hysterophorus plants are infected by a phytoplasma. Partial sequencing of 16S rRNA (GenBank accession: LT671581 and LT671583) and virtual restriction fragment length polymorphism confirmed that the phytoplasma associated with both plant species had the greatest homology to 16SrII-D subgroup. Disease was successfully transmitted by grafting and leafhopper Orosius albicinctus in tomato plants. This is the first report of natural occurrence of 16SrII-D phytoplasma in tomatoes and a weed P. hysterophorus in Pakistan.     

Rapid Detection by Genomic Amplification of Loofah Witches' Broom Phytoplasma in Leafhopper Vectors

Loofah (Luffa cylindrical) is a common vegetable crop in Taiwan and other Asian countries. Reported here is a novel rapid approach for detecting loofah witches’ broom (LfWB) phytoplasma in single leafhoppers. Field samples of suspected diseased plants and potential vectors from southern Taiwan were processed to test for the presence of the LfWB phytoplasmas using both strain‐specific DNA hybridization (DH) and polymerase chain reaction (PCR) assays. The commonest pathogen causing loofah disease in southern Taiwan is LfWB phytoplasma. Leafhoopers collected at nine locations near LfWB‐infected plants were found to be positive for LfWB by PCR / DH at an incidence of 28.5–40.0%. Of the different leafhopper species tested, only Hishimonus concavus was positive for LfWB, suggesting that H. concavus is a natural vector of LfWB in Taiwan. Using our proposed primers in this PCR assay, a single LfWB‐infected leafhopper can be detected rapidly and directly.