Identification of aster yellows phytoplasma in garlic and green onion by PCR-based methods

In the summer of 1999, typical yellows-type symptoms were observed on garlic and green onion plants in a number of gardens and plots around Edmonton, Alberta, Canada. DNA was extracted from leaf tissues of evidently healthy and infected plants. DNA amplifications were conducted on these samples, using two primer pairs, R16F2n/R2 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. DNA samples of aster yellows (AY), lime witches'-broom (LWB) and potato witches'-broom (PWB) phytoplasmas served as controls and were used to determine group relatedness. In a direct polymerase chain reaction (PCR) assay, DNA amplification with universal primer pair R16F2n/R2 gave the expected amplified products of 1.2 kb. Dilution (1/40) of each of the latter products were used as template and nested with specific primer pair R16(1)F1/R1. An expected PCR product of 1.1 kb was obtained from each phytoplasma-infected garlic and green onion samples, LWB and AY phytoplasmas but not from PWB phytoplasma. An aliquot from each amplification product (1.2 kb) with universal primers was subjected to PCR-based restriction fragment length polymorphism (RFLP) to identify phytoplasma isolates, using four restriction endonucleases (AluI, KpnI, MseI and RsaI). DNA amplification with specific primer pair R16(1)F1/R1 and RFLP analysis indicated the presence of AY phytoplasma in the infected garlic and green onion samples. These results suggest that AY phytoplasma in garlic and green onion samples belong to the subgroup 16Sr1-A.     

Molecular Characterization of Aster Yellows Phytoplasma Associated with Valerian and Sowthistle Plants by PCR–RFLP Analyses

In Alberta, Canada, valerian grown for medicinal purposes and sowthistle, a common weed, showed typical aster yellows symptoms. Molecular diagnosis was made using a universal primer pair (P1 / P7) designed to amplify the entire 16S rRNA gene and the 16 / 23S intergenic spacer region in a direct polymerase chain reaction (PCR) assay. This primer pair amplified the DNA samples from valerian and sowthistle and reference controls (AY‐27, CP, PWB, AY of canola, LWB). They produced the expected PCR products of 1.8 kb, which were diluted and used as templates in a nested PCR. Two primer pairs R16F2n / R2 and P3 / P7 amplified the DNA templates giving PCR products of 1.2 and 0.32 kb, respectively. No PCR product was obtained with either set of primers and DNA isolated from healthy plants. Restriction fragment length polymorphism (RFLP) was used to analyse the partial 16S rDNA sequences (1.2 kb) of all phytoplasma DNA samples after restriction with four endonucleases (AluI, HhaI, MseI and RsaI). The restriction patterns of these strains were found to be identical with the RFLP pattern of the AY phytoplasma reference control (AY‐27 strain). Based on the RFLP data, the two strains are members of subgroup A of the AY 16Sr1 group. We report here the first molecular study on the association of AY phytoplasmas with valerian and sowthistle plants.     

Detection of aster yellows phytoplasma in false flax based on PCR and RFLP

False flax (Camelina sativa L.) plants were found to be infected with a yellows-type disease caused by a phytoplasma in experimental plots at the Edmonton Research station. Alberta, Canada. Typical phytoplasmas were detected in the phloem cells in ultrathin sections from leaf midrib tissues examined by electron microscopy. These observations were supported by polymerase chain reaction (PCR) using two primer pairs, R16 F2n/R2 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. Aster yellows (AY) and potato witches'-broom (PWB) phytoplasma DNA samples served as controls and were used to study group relatedness. In a direct PCR assay, DNA amplification with universal primer pair R16F2n/R2 gave the expected PCR products of 1.2 kb. Based on a nested-PCR assay using the latter PCR products as templates, and a specific primer pair, R16(1)F1/R1, designed on the basis of AY phytoplasma rDNA sequences, a PCR product of 1.1 kb was obtained from each phytoplasma-infected false flax and AY sample, but not from PWB phytoplasma and healthy controls. DNA amplification with specific primer pair R16(1)F1/R1 and restriction fragment length polymorphism indicated the presence of AY phytoplasma in the infected false flax sample. This is the first reported characterization of AY phytoplasma in false flax.     

Aster Yellows Phytoplasma Identified in Scentless Chamomile by Microscopical Examinations and Molecular Characterization

Scentless chamomile (Matricaria perforata Mérat) plants were commonly found infected with a yellows-type disease caused by phytoplasma in several fields in Alberta, Canada. Typical phytoplasmas were detected in the phloem cells in ultrathin sections from leaf, stem, root and flower petiole tissues examined by electron microscopy. Application of 4′6-diamidino-2-phenylindole- 2HCl (DAPI) staining techniques confirmed the presence of the phytoplasma in these tissues. These observations were supported by polymerase chain reaction (PCR) assays, using two primer pairs, P1/P6 and R16(1)F1/R1, derived from phytoplasma rDNA sequences. Aster yellows and potato witches′-broom (PWB) DNA phytoplasma samples served as positive controls and were used to study group relatedness. In a direct PCR assay, DNA amplification with universal primer pair P1/P6 gave the expected PCR products of 1.5 kb. Based on a nested-PCR assay using the latter PCR products, as templates, and a specific primer pair R16(1)F1/R1 designed on the basis of AY phytoplasma rDNA sequences, a PCR product of 1.1 kb was obtained from each phytoplasma-infected chamomile and AY samples but not from PWB phytoplasma and healthy chamomile controls. DNA amplification with specific primer pair R16(1)F1/R1 and restriction fragment length polymorphism indicated the presence of AY phytoplasma in the infected scentless chamomile sample.     

The Association of Phytoplasma with Stunting, Leaf Necrosis and Witches' Broom Symptoms in Magnolia Plants

In 1999–2000 a severe disease was observed on plants of four Magnolia spp. cultivated in a commercial nursery in Poland. Affected plants showed a progressive loss of vigour, were stunted, and had severely malformed leaves, leaf necrosis and witches' broom. Phytoplasma was detected in magnolias with severe symptoms and in dodder-inoculated Catharanthus roseus seedlings by nested polymerase chain reaction (PCR) assay with primer pair R16F1/R0 followed by universal (rA/fA) and group specific (R16(I)F1/R1) primer pairs which amplified a fragment of phytoplasma 16S rDNA. The PCR products (560 bp or 1.1 kb) of all samples used for restriction fragment length polymorphism analysis after digestion with endonuclease enzymes Alu I and Mse I produced the same profile which corresponded to that of an aster yellows phytoplasma reference strain. Phytoplasma DNA was detected throughout the growing season in roots, stems and young but not mature leaves. Electron microscope examination of the ultra-thin sections of the leaf and stem of diseased magnolias showed collapsed and degenerated sieve tube elements with wall thickening. The reduced lumen of these sieve elements contained numerous vesicles and membrane-bound structures, but no typical phytoplasma cells. This is the first report of aster yellows phytoplasma in magnolia identified by molecular assays.     

Molecular Evidence for the Presence of Aster Yellows-Related Phytoplasmas in Lilies with Leaf Scorch and Flower Virescence

Severe leaf scorch symptoms occurred on oriental lily hybrids cv. Woodriff's Memory cultivated in two commercial greenhouses in Poland. Symptoms included leaf necrosis and malformation, flower bud abscission and flower virescence, distortion and abortion. Naturally infected lily plants with severe symptoms in 1999 had retarded growth and leaf chlorosis and they failed to flower the following year. The presence of phytoplasmas in diseased lilies was demonstrated using nested polymerase chain reaction (PCR) assays with universal and 16SrI group specific primer pairs that amplified the phytoplasma 16S rDNA fragment. The PCR products (1.1 kb) of all samples used for restriction fragment length polymorphism analysis had the same restriction profiles after digestion with endonucleases Alu I and Mse I. The restriction profiles of phytoplasma DNA from these plants corresponded to those of an aster yellows phytoplasma reference strain.     

Detection of Phytoplasma Infection in Rose, with Degeneration Symptoms

In 1998 a severe disease was observed on rose cvs. 'Patina', 'Papillon' and 'Mercedes' cultivated in a commercial greenhouse in Poland. The symptoms included stunted growth, bud proliferation, leaf malformation and deficiency of flower buds. Sporadically some plants yielded flower buds transformed into big-bud structures and degenerated flowers. The presence of phytoplasma in roses with severe symptoms as well as in recovered plants and Catharanthus roseus experimentally infected by grafting and via dodder was demonstrated by nested polymerase chain reaction assay with primers pair R16F2/R2 or R16F1/R0 and R16(I)F1/R1 amplifying phytoplasma 16S rDNA fragment. The polymerase chain reaction products (1.1 kb) used for restriction fragment length polymorphism analysis after digestion with endonuclease enzymes Alu I and Mse I produced the same restriction profiles for all samples. The restriction profiles of phytoplasma DNA from these plants corresponded to those of an aster yellows phytoplasma reference strain. Electron microscope examination of the ultra-thin sections of the stem showed wall thickenings of many sieve tubes of the diseased roses and single phytoplasma cells within a sieve element of the phloem of experimentally infected periwinkles. This paper is the first report on aster yellows phytoplasma in rose identified at a molecular level.     

Detection and molecular characterization of an aster yellows phytoplasma in poker statice and Queen Anne's lace in Alberta, Canada

Queen Anne's lace and poker statice plants were found with a yellows-type disease with typical phytoplasma symptoms in an experimental farm near Brooks, Alberta in 1996. Phytoplasma bodies were detected by transmission electron microscopy in phloem cells of symptomatic plants, but not in healthy plants. The presence of a phytoplasma was confirmed by analysis with the polymerase chain reaction. Using a pair of universal primer sequences derived from phytoplasma 16S rRNA, an amplified product of the expected size (1.2 kb) was observed in samples from infected plants, but not in asymptomatic plants. Sequence analysis of the PCR products from the 16S/23S rDNA intergenic spacer region indicated that the two phytoplasma isolates in Queen Anne's lace and poker statice are genetically closely related to the western aster yellows phytoplasma.     

Niger Seed (Guizotia abyssinica), a New Host of ‘Candidatus Phytoplasma asteris’ in Iran

Shrubs of niger seed with phyllody and internode elongation symptoms suggestive of phytoplasma infections occurred in the central regions of Iran. Phytoplasma was detected by polymerase chain reaction (PCR) and nested PCR amplifications using phytoplasma universal primer pairs P1/P7 and R16F2n/R16R2. Using aster yellows group–specific primer pair rp(I)F1A/rp(I)R1A, a fragment of 1212 bp of the rp genes was amplified from DNA samples of infected plants. Random fragment length polymorphism (RFLP) analyses of R16F2n/R16R2‐amplified products using the CfoI restriction enzyme confirmed that Iranian niger seed phyllody phytoplasma is associated with aster yellows group phytoplasmas. Sequence analyses of the partial rp genes fragment indicated that the Iranian niger seed phyllody phytoplasma, which was collected from central regions of Iran, is related to ‘Candidatus Phytoplasma asteris’. This is the first report of a phytoplasma infecting the niger seed plant.     

Detection and Molecular Characterization of a Phytoplasma Associated with Big Bud Disease of Tomatoes in Jordan

Tomato big bud was detected for the first time in tomato plants (Lycopersicon esculentum Mill.) in the eastern region (Al‐Mafraq) of Jordan. Infected plants showed proliferation of lateral shoots, hypertrophic calyxes and greening of flower petals. The presence of phytoplasmas in diseased tomato plants was demonstrated using polymerase chain reaction (PCR) assays. The amplified DNAs yielded products of 1.8 kb (primer pair P1/P7) and 1.2 kb (primer pair R16F2/R2) by direct and nested‐PCR, respectively. DNA from tomato isolates T1 and T2 could not be amplified in the nested‐PCR assays when the aster yellow‐specific primer pair R16(1)F1/R1 was used, suggesting that the phytoplasma in these isolates is not genetically related to the 16SrI (aster yellows) group. After restriction fragment length polymorphism (RFLP) analyses, using four endonuclease enzymes (HhaI, RsaI, AluI and Bsp143I) similar patterns were formed among the digested 1.2 kb PCR products of two tomato isolates suggesting that both isolates belonged to the same phytoplasma. Compared with the RFLP profile of the reference strains, no difference in the digestion pattern was found between the tomato isolates and that of the catharanthus phyllody agent from Sudan, indicating that the phytoplasma belongs to 16SrDNA VI (clover proliferation) group.     

Identification of Elm Yellows Phytoplasma in Plum Trees in China

Plum plants (Prunus cerasifera Ehrh) with small and rolled leaves resembling symptoms of phytoplasma infection were observed during 2008 and 2009 in the ornamental garden of Northwest A&F University (Republic of China). Nested polymerase chain reaction (PCR) using a combination of phytoplasma‐specific universal primer pairs (R16F2m/R16R1m‐R16F2n/R16R2) amplified 16S rDNA with the expected size (1.2 kb) from all samples of symptomatic plum plants. Sequencing results and restriction fragment length polymorphism (RFLP) analysis of the 1248 bp R16F2n/R16R2 products showed that the phytoplasma belongs to group 16SrV. Phylogenetic analysis showed that the phytoplasma had a close relation to JWB phytoplasma. This is, we believe, the first report of elm yellows phytoplasma infecting plum plants in China.     

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.     

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.     

Occurrence,Molecular Characterization and Vector Transmission of a Phytoplasma Associated with Rapeseed Phyllody in Iran

Symptoms of rapeseed phyllody were observed in rapeseed fields of Fars, Ghazvin, Isfahan, Kerman and Yazd provinces in Iran. Circulifer haematoceps leafhoppers testing positive for phytoplasma in polymerase chain reaction (PCR) successfully transmitted a rapeseed phyllody phytoplasma isolate from Zarghan (Fars province) to healthy rapeseed plants directly after collection in the field or after acquisition feeding on infected rapeseed in the greenhouse. The disease agent was transmitted by the same leafhopper from rape to periwinkle, sesame, stock, mustard, radish and rocket plants causing phytoplasma‐type symptoms in these plants. PCR assays using phytoplasma‐specific primer pair P1/P7 or nested PCR using primers P1/P7 followed by R16F2n/R2, amplified products of expected size (1.8 and 1.2 kbp, respectively) from symptomatic rapeseed plants and C. haematoceps specimens. Restriction fragment length polymorphism analysis of amplification products of nested PCR and putative restriction site analysis of 16S rRNA gene indicated the presence of aster yellows‐related phytoplasmas (16SrI‐B) in naturally and experimentally infected rapeseed plants and in samples of C. haematoceps collected in affected rapeseed fields. Sequence homology and phylogenetic analysis of 16S rRNA gene confirmed that the associated phytoplasma detected in Zarghan rapeseed plant is closer to the members of the subgroup 16SrI‐B than to other members of the AY group. This is the first report of natural occurrence and characterization of rapeseed phyllody phytoplasma, including its vector identification, in Iran.     

Heteroduplex mobility assay detects DNA mutations for differentiation of closely related phytoplasma strains

We present the first use of DNA heteroduplex mobility assay (HMA) to detect the point mutations including substitutions and deletions/insertions in 16S rDNA of aster yellows phytoplasma (AY27) and to differentiate phytoplasmas collected from field samples of clover proliferation (CP) and alfalfa witches'-broom (AWB). The phytoplasmal 16S rDNA fragment was amplified from AY27 by polymerase chain reaction (PCR) and cloned into a plasmid vector. The cloned DNA fragment was subjected to in vitro mutation to produce 1- to 4-base substitutions and 1- to 3-base deletions. The mutated 16S rDNA fragments were analyzed by HMA. The results showed that a single two-base substitution or a single-base deletion/insertion in the 529 bp DNA fragment was directly detected and that a DNA divergence at a level of as low as 0.2% was detectable by HMA. Heteroduplex mobilities were affected by the number and composition of the phytoplasma DNA bases in mismatches or gaps and were proportional to the degree of DNA divergences. Gaps caused greater retardation in heteroduplex mobility than mismatches did. HMA was highly sensitive in detecting the mixed infections of phytoplasmas. In analyses of CP and AWB field samples collected in Alberta, two CP and one AWB phytoplasma isolates were differentiated from others by HMA but not by restriction fragment length polymorphism (RFLP). Therefore, HMA provides a simple, rapid, highly sensitive and analytical method to detect and estimate the genetic divergence of phytoplasmas when other methods such as RFLP are not readily applicable.     

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.     

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 ~ 1.3 kb 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 1.2 kb 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 vegetables and 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 of 16SrVI-D subgroup of phytoplasmas infecting vegetable and other important agricultural crops.     

‘Candidatus Phytoplasma asteris’ and ‘Candidatus Phytoplasma aurantifolia’, New Phytoplasma Species Infecting Apple Trees in Iran

During several surveys in extensive areas in central Iran, apple trees showing phytoplasma diseases symptoms were observed. PCR tests using phytoplasma universal primer pairs P1A/P7A followed by R16F2n/R16R2 confirmed the association of phytoplasmas with symptomatic apple trees. Nested PCR using 16SrX group‐specific primer pair R16(X)F1/R1 and aster yellows group‐specific primer pairs rp(I)F1A/rp(I)R1A and fTufAy/rTufAy indicated that apple phytoplasmas in these regions did not belong to the apple proliferation group, whereas aster yellows group‐related phytoplasmas caused disease on some trees. Restriction fragment length polymorphism (RFLP) analyses using four restriction enzymes (HhaI, HpaII, HaeIII and RsaI) and sequence analyses of partial 16S rRNA and rp genes demonstrated that apple phytoplasma isolates in the centre of Iran are related to ‘Ca. Phytoplasma asteris’ and ‘Ca. Phytoplasma aurantifolia’. This is the first report of apples infected with ‘Ca. Phytoplasma asteris’ in Iran and the first record from association of ‘Ca. Phytoplasma aurantifolia’ with apples worldwide.     

长春花黄化植原体（PY）株系的检测与鉴定

植原体 (Ｐｈｙｔｏｐｌａｓｍａ) (原称类菌原体Ｍｙｃｏｐｌａｓｍａ ｌｉｋｅＯｒｇａｎｉｓｍ ,简称ＭＬＯ)是一类无细胞壁、存在于植物筛管细胞内的原核生物。植原体自 1 967年被日本学者土居养二首次发现后 ,迄今为止 ,世界上报道的植物植原体病害多达 30 0余种 ,早期对植原体的鉴定主要是通过生物学特性 ,如症状特征、与昆虫介体的相互关系等进行的。这些方法费时费力 ,结果往往也不是很可靠。 80年代 ,随着血清学、分子探针以及ＰＣＲ技术的发展应用 ,为植原体的检测提供了一种相对简单、灵敏、可靠的方法。通过对 1 6ＳｒＲＮＡ基…