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.     

Association of a phytoplasma with a yellow leaf syndrome of sugarcane in Africa

Evidence is presented for the association of a phytoplasma, provisionally named sugarcane yellows phytoplasma (ScYP), in sugarcane affected by a yellow leaf syndrome. The phytoplasma was consistently detected in leaves of more than 40 varieties from eight African countries. It was present in all symptomatic as well as some asymptomatic field grown cane samples but not in plants grown from true seed, and it was also observed in phloem sieve tubes by transmission electron microscopy. Phytoplasma 16S rDNA was confirmed by PCR, and restriction fragment analysis using Rsal and Haelll confirmed that PCR-amplified products were of phytoplasma rather than of plant or of other pathogen origin. Sequences obtained from the intergenic spacer region, between the 16S and 23S rDNA genes, confirmed the identity of the phytoplasma as belonging to the western X group of phytoplasmas.     

Artemisia vulgaris,a new host of 16SrV-C phytoplasma related strains infecting black alder in Poland

Yellowing of leaf tissue and strongly deformed shoots were observed in common mugwort (Artemisia vulgaris L.) growing in a nature reserve in Southern Poland. Similar foliage chlorosis together with abnormal shoot proliferation was noticed on alder tree (Alnus glutinosa Gaertn.) growing next to the common mugwort. DNA specific fragments coding 16S rRNA and ribosomal proteins (rp) were amplified from mugwort and alder samples using direct and nested PCR (Polymerase Chain Reaction) assays. Phylogenetic relationships inferred from 16S and rps3 genes indicated that strains infecting mugwort and alder were most closely related to phytoplasmas of subgroups 16SrV-C and 16SrV-D. Based on the restriction fragment length polymorphism (RFLP) analysis of the 16S rDNA, the investigated phytoplasma strains were classified to subgroup 16SrV-C. Two sequence variants of the rps3 gene which differed by a single nucleotide were detected in all analysed samples by pairwise analysis of the aligned reads. Taking into account that this single-nucleotide polymorphism (SNP) occurs among 16SrV-C and 16SrV-D related phytoplasmas and that the phytoplasmas have a single copy of rp operon, we concluded that each plant species was infected by two distinct, closely related phytoplasma strains. To the best of our knowledge, this is the first report of group 16SrV-C related phytoplasmas infecting common mugwort worldwide, adding a new host species that is possibly linked to the spread of the alder pathogen in Eastern Europe. Although alder yellows phytoplasma has been frequently found in Europe, this is the first detection of phytoplasmas associated with alder in Poland.     

Detection and identification of ‘Candidatus Phytoplasma asteris’ in Brassica rapa subsp. pekinensis in Poland

Severe growth abnormalities, including leaf yellowing, sprout proliferation and flower virescence and phyllody, were found on Brassica rapa subsp. pekinensis plants in Poland. The presence of phytoplasma in naturally infected plants was demonstrated by polymerase chain reaction assay employing phytoplasma universal P1/P7 followed by R16F2n/R16R2 primer pairs. The detected phytoplasma was identified using restriction fragment length polymorphism analysis (RFLP) of the 16S rRNA gene fragment with AluI, HhaI, MseI and RsaI endonucleases. After enzymatic digestion, all tested samples showed restriction pattern similar to that of ‘Candidatus phytoplasma asteris’. Nested PCR‐amplified products, obtained with primers R16F2n/R16R2, were sequenced. Sequences of the 16S rDNA gene fragment of analysed phytoplasma isolates were nearly identical. They revealed high nucleotide sequence identity (>98%) with corresponding sequences of other phytoplasma isolates from subgroup 16SrI‐B, and they were classified as members of ‘Candidatus phytoplasma asteris’. This is the first report of the natural occurrence of phytoplasma‐associated disease in plants of Chinese cabbage.     

A new phytoplasma associated with little leaf disease in azalea: multilocus sequence characterization reveals a distinct lineage within the aster yellows phytoplasma group

An azalea little leaf (AzLL) disease characterised by abnormally small leaves, yellowing and witches'‐broom growth symptoms was observed in suburban Kunming, southwest China. Transmission electron microscopic observations of single‐membrane‐bound, ovoid to spherical bodies in phloem sieve elements of diseased plants and detection of phytoplasma‐characteristic 16S rRNA gene sequence in DNA samples from diseased plants provided evidence linking the disease to infection by a phytoplasma. Results from restriction fragment length polymorphism, phylogenetic and comparative structural analyses of multiple genetic loci containing 16S rRNA, rpsS, rplV, rpsC and secY genes indicated that the AzLL phytoplasma represented a distinct, new 16Sr subgroup lineage, designated as 16SrI‐T, in the aster yellows phytoplasma group. The genotyping also revealed that the AzLL phytoplasma represented new rp and secY gene lineages [rp(I)‐P and secY(I)‐O, respectively]. Phylogenetic analyses of secY and rp gene sequences allowed clearer distinctions between AzLL and closely related strains than did analysis of 16S rDNA.     

Multilocus genotyping of a ‘Candidatus Phytoplasma aurantifolia’‐related strain associated with cauliflower phyllody disease in China

A new cauliflower disease characterised by the formation of leaf‐like inflorescences and malformed flowers occurred in a seed production field located in Yunnan, a southwest province of China. Detection of phytoplasma‐characteristic 16S rRNA gene sequences in DNA samples from diseased plants linked the cauliflower disease to phytoplasmal infection. Results from phylogenetic and virtual restriction fragment length polymorphism analyses of the 16S rRNA gene sequence indicated that the cauliflower‐infecting agent is a ‘Candidatus Phytoplasma aurantifolia’‐related strain and is a new member of the peanut witches'‐broom phytoplasma group, subgroup A (16SrII‐A). Multilocus genotyping showed close genetic relationship between this cauliflower phytoplasma and a broad host range phytoplasma lineage found only in East Asia thus far. Molecular markers present in the secY and rp loci distinguished this phytoplasma from other members of the subgroup 16SrII‐A.     

Sequence Analysis of 16S rRNA and secA Genes Confirms the Association of 16SrI‐B Subgroup Phytoplasma with Oil Palm (Elaeis guineensis Jacq.) Stunting Disease in India

During January 2010, severe stunting symptoms were observed in clonally propagated oil palm (Elaeis guineensis Jacq.) in West Godavari district, Andhra Pradesh, India. Leaf samples of symptomatic oil palms were collected, and the presence of phytoplasma was confirmed by nested polymerase chain reaction (PCR) using universal phytoplasma‐specific primer pairs P1/P7 followed by R16F2n/R16R2 for amplification of the 16S rRNA gene and semi‐nested PCR using universal phytoplasma‐specific primer pairs SecAfor1/SecArev3 followed by SecAfor2/SecArev3 for amplification of a part of the secA gene. Sequencing and BLAST analysis of the ～1.25 kb and ～480 bp of 16S rDNA and secA gene fragments indicated that the phytoplasma associated with oil palm stunting (OPS) disease was identical to 16SrI aster yellows group phytoplasma. Further characterization of the phytoplasma by in silico restriction enzyme digestion of 16S rDNA and virtual gel plotting of sequenced 16S rDNA of ～1.25 kb using iPhyClassifier online tool indicated that OPS phytoplasma is a member of 16SrI‐B subgroup and is a ‘Candidatus Phytoplasma asteris’‐related strain. Phylogenetic analysis of 16S rDNA and secA of OPS phytoplasma also grouped it with 16SrI‐B. This is the first report of association of phytoplasma of the 16SrI‐B subgroup phytoplasma with oil palm in the world.     

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.     

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.     

Characterisation of phytoplasma (16SrVI) associated with little leaf disease of Portulaca grandiflora – An in silico analysis

A new severe little leaf disease was observed on P. grandiflora, popular as Moss-rose Purslane, widely grown in temperate zones. Characteristic symptoms, ultrastructural studies, antibiotic response and amplification of 16S ribosomal DNA fragments (about 1.5 kb) by polymerase chain reaction (PCR) from infected samples, suspect the involvement of phytoplasma as a pathogen. Nested PCR product, 1.2 kb, with primer pairs R16F2n/R16R2 used for cloning and sequencing. Comparision of the 16S rRNA gene sequences showed that the causal, PLL phytoplasma, is very close (98%) to Indian brinjal little leaf (EF186820) and “Candidatus Phytoplasma trifolii” (AY390261), 16SrVI group phytoplasmas, previously reported from India and Canada respectively. Here, the status of PLL (EF651786) is verified by computer-simulated restriction fragment length polymorphism analysis of 16S rRNA genes of the F2n/R2 sequences of closely related strains of the 16SrVI group using 17 restriction enzymes.     

First Report of Aster Yellows Phytoplasma in Goldenrain Tree (Koelreuteria paniculata) in Korea

Aster yellows phytoplasma was detected for the first time in goldenrain tree (Koelreuteria paniculata) growing in Sinpyeong‐myeon, Jeollabuk‐do, South Korea. DNA was extracted from the infected leaf samples and part of the 16S rDNA, rp operon and tuf gene were amplified using R16F2n/R2 and gene‐specific primers. The sequence analysis showed that the phytoplasma was closely related (99%) to members of the Aster Yellows (AY) group, and belonging to 16Sr I, subgroup B. Moreover, the 16S rDNA sequences of the isolate showed 88–96% identity with members of other 16Sr and undesignated groups. Based on the sequence identity and phylogenetic studies, it was confirmed that phytoplasma infecting goldenrain tree in South Korea belongs to the AY group.     

Aster Yellows Subgroup 16SrI‐C Phytoplasma in Rhododendron hybridum

Symptoms of unknown aetiology on Rhododendron hybridum cv. Cunningham's White were observed in the Czech Republic in 2010. The infected plant had malformed leaves, with irregular shaped edges, mosaic, leaf tip necrosis and multiple axillary shoots with smaller leaves. Transmission electron microscopy showed phytoplasma‐like bodies in phloem cells of the symptomatic plant. Phytoplasma presence was confirmed by polymerase chain reaction using phytoplasma‐specific, universal and group‐specific primer pairs. Restriction fragment length polymorphism analysis of 16S rDNA enabled classification of the detected phytoplasma into the aster yellows subgroup I‐C. Sequence analysis of the 16S‐23S ribosomal operon of the amplified phytoplasma genome from the infected rhododendron plant (1724 bp) confirmed the closest relationship with the Czech Echinacea purpurea phyllody phytoplasma. These data suggest Rhododendron hybridum is a new host for the aster yellows phytoplasma subgroup 16SrI‐C in the Czech Republic and worldwide.     

First Report of a 16SrI‐C Phytoplasma Infecting Celery (Apium graveolens) with Stunting,Bushy Top and Phyllody in the Czech Republic

Apium graveolens L. plants showing stunting, purplish/whitening of new leaves, flower abnormalities and bushy tops were observed in South Bohemia (Czech Republic) during 2011 and 2012. Transmission electron microscopy observations showed phytoplasmas in phloem sieve tube elements of symptomatic but not healthy plants. Polymerase chain reactions with universal and group‐specific phytoplasma primers followed by restriction fragment length polymorphism analyses and sequencing of 16S rDNA enabled classification of the detected phytoplasmas into the aster yellows group, ribosomal subgroup 16SrI‐C. Identical analyses of the ribosomal protein genes rpl22 and rps3 were used for further classification and revealed affiliation of the phytoplasmas with the rpIC subgroups. This is the first report of naturally occurring clover phyllody phytoplasma in A. graveolens in both the Czech Republic and worldwide.     

Surveys reveal the occurrence of phytoplasmas in plants at different geographical locations in Peru

Two independent surveys were performed in Peru during February and November 2007 to detect the presence of phytoplasmas within any crops showing symptoms resembling those caused by phytoplasmas. Molecular identifications and characterisations were based on phytoplasma 16S and 23S rRNA genes using nested PCR and terminal restriction fragment length polymorphism (T‐RFLP). The surveys indicated that phytoplasmas were present in most of the locations sampled in Peru in both cultivated crops, including carrots, maize, native potatoes, improved potato, tomato, oats, papaya and coconut, and in other plants such as dandelion and the ornamental Madagascar periwinkle (Catharanthus roseus). Phylogenetic analysis of the sequences confirmed that while most of the isolates belong to the 16SrI aster yellows group, which is ubiquitous throughout other parts of South America, one isolate from potato belongs to the 16SrII peanut witches’ broom group, and one isolate from tomato and one from dandelion belong to the 16SrIII X‐disease group. The use of T‐RFLP was validated for the evaluation of phytoplasma‐affected field samples and provided no evidence for mixed infection of individual plants with more than one phytoplasma isolate. These data represent the first molecular confirmation of the presence of phytoplasmas in a broad range of crops in Peru.     

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.     

Detection and Molecular Characterization of a 16SrII‐A* Phytoplasma in Grapefruit (Citrus paradisi) with Huanglongbing‐like Symptoms in China

In the year 2010, in a survey in Guangxi Province, China, to detect and characterize phytoplasmas in a huanglongbing (HLB)‐infected grapefruit (Citrus paradisi) orchard, 87 leaf samples with symptoms of blotchy mottle were collected from symptomatic grapefruit trees, and 320 leaf samples from symptomless trees adjacent to the symptomatic trees. Nested polymerase chain reaction (PCR) using universal phytoplasma primer set P1/P7 followed by primer set fU5/rU3 identified 7 (8.0%) positive samples from symptomatic samples but none from symptomless samples. Of the 87 symptomatic samples, 77 (88.5%) were positive for ‘Candidatus Liberibacter asiaticus’ and 5 for both phytoplasma and ‘Ca. L. asiaticus’. Sequence analysis indicated that seven 881‐bp amplicons, amplified by nested phytoplasma primer sets P1/P7 and fU5/rU3, shared 100.0% sequence identity with each other. Genome walking was then performed based on the 881 bp known sequences, and 5111 bp of upstream and downstream sequences were obtained. The total 5992 bp sequences contained a complete rRNA operon, composed of a 16S rRNA gene, a tRNA^Ile gene, a 23S rRNA gene and a 5S rRNA gene followed by eight tRNA genes. Phylogenetic analysis and virtual restriction fragment length polymorphism analysis confirmed the phytoplasma was a variant (16SrII‐A*) of phytoplasma subgroup 16SrII‐A. As phytoplasmas were only detected in blotchy‐mottle leaves, the 16SrII‐A* phytoplasma identified was related to HLB‐like symptoms.     

Detection and Characterization of Phytoplasma and Sugarcane Yellow Leaf Virus Associated with Leaf Yellowing of Sugarcane

Leaves from sugarcane were collected from Egyptian plantation fields and tested for phytoplasma (Sugarcane yellows phytoplasma, SCYP) and Sugarcane yellow leaf virus (SCYLV) using nested PCR (with different primers) and RT‐PCR, respectively. These results showed significant differences in the amplification of the PCR assays. The primer MLO‐X/MLO‐Y, which amplified the 16S‐23S rDNA spacer region, was the most precise to detect the phytoplasma in sugarcane plants. Sequencing and restriction fragment length polymorphism analysis revealed that all tested phytoplasmas belonged to the 16SrI (aster yellows phytoplasma) group, with the exception of cultivar G84‐47 belonged to the 16SrXI (Rice yellow dwarf phytoplasma) group. Three Egyptian sugarcane cultivars were phytoplasma free. Phylogenetic analyses of 34 screened accessions of 16S ribosomal DNA gene sequences of Candidatus phytoplasma including the ones collected from Egypt used in this study and those extracted from GenBank showed that they split into two distinct clusters. The phylogenetic analyses indicated that these phytoplasmas are closely related and share a common ancestor. All tested Egyptian sugarcane plants were infected by SCYLV with the exception of cultivar Phil‐8013 which was virus free.     

Electron Microscopy and Molecular Characterization of Phytoplasmas Associated with Little Leaf Disease of Brinjal (Solanum melongena L.) and Periwinkle (Catharanthus roseus) in Bangladesh

In Bangladesh little leaf disease was observed in brinjal ( Solanum melongena L.) and in periwinkle ( Catharanthus roseus ). Phloem-inhabiting phytoplasmas were consistently detected in both species of diseased plants using transmission electron microscopy (TEM) and polymerase chain reaction (PCR) techniques. The shape, size and within-tissue distribution of phytoplasmas appears to be similar in both hosts. Furthermore, the molecular characterization and identifications of observed phytoplasmas were carried out based on restriction fragment length polymorphism (RFLP) patterns of PCR-amplified products (1200 bp) using phytoplasma-specific universal primers and sequencing analysis of both 16S ribosomal DNA (rDNA) and intergenic spacer region (ISR) of 16S-23S rDNA phytoplasma genes. The patterns of RFLP analysis with seven restriction enzymes exhibited a similar pattern for both phytoplasma strains. The sequence homology between these two strains showed 100% similarity based on 16S rDNA and 16S-23S ISR. Therefore, in Bangladesh the causal agents of brinjal little leaf (BLL-Bd) and periwinkle little leaf (PLL-Bd) are probably the same or closely related phytoplasma strains. These strains, are very close or identical to the strain of brinjal little leaf phytoplasma in India (BLL-In), belonging to the clover proliferation group (Lee et al., Int. J. Syst. Bacteriol. 48, 1153–1169, 1998; Seemuller et al., J. Plant Pathol. 80, 3–26, 1998).     

Association of ‘Candidatus Phytoplasma cynodontis’ with Bermuda grass white leaf disease and its new hosts in Qassim province,Saudi Arabia

Typical symptoms of phytoplasma such as whitening of the leaves, shortening of the stolons on Bermuda grass, variegated leaves, yellows, stunting, little leaves and yellows on Giant reed, Cooba and sand olive shrub were observed in Qassim province, Saudi Arabia, during the autumn season of 2015. When tested for phytoplasma by universal primers P1/P7 followed by R16mF2/R16mR2, products of approximately 1400?bp (as expected) were amplified from 16 plants with symptoms but not from symptomless plants. Based on sequencing, phylogenetic analysis and virtual restriction fragment length polymorphism patterns of the 16S rDNA F2nR2 fragments of seven Qassim phytoplasma isolates, bermuda grass isolates 170, 175 and 177, giant reed isolate 180, sand olive isolates 181 and 182 and cooba isolate 185, the associated phytoplasma was identified as a member of ‘Candidatus Phytoplasma cynodontis’ which belong to the 16SrXIV-A subgroup. The 16S rDNA gene sequences of seven Qassim phytoplasma isolates exhibited over 99.2% identity with members of ‘Ca. Phytoplasma cynodontis’ group of phytoplasmas. This is the first report of characterization of ‘Ca. phytoplasma cynodonties’ (16SrXIV) associated with Cynodon dactylon in Saudi Arabia and its new hosts, Dodonaea angustifolia, Arundo donax and Acacia salicia.     

Characterisation and phylogeny of a phytoplasma inducing sandal spike disease in sandal (Santalum album)

Sandal (Santalum album) is an industrially important forest species in India, where it is devastated by sandal spike (SAS) disease. Diseased S. album trees show characteristic witches’ broom symptoms suspected to be caused by phytoplasma. Since the first report of occurrence of this disease at the end of 19th century, studies mainly have been carried out to detect SAS phytoplasma through various approaches. The causative agent, however, has remained poorly characterised at a molecular level. The present investigation was aimed to characterise the pathogen at this level. In nested PCR, a 1.4‐kb 16S rDNA fragment was amplified and analysed by restriction fragment length polymorphism using 17 restriction enzymes. The patterns were identical to those of strains AY1 and APh of the aster yellows subgroup 16SrI‐B, except for BfaI, which gave a different pattern. After cloning and sequencing, a phylogenetic analysis revealed the closest relationship to aster yellows subgroup 16SrI‐B members. Nucleotide sequence identity ranged from 99.2% to 99.5% with this subgroup. On the basis of these results, the SAS phytoplasma was classified as a member of subgroup 16SrI‐B.