Detection and molecular characterization of phytoplasmas associated with vegetable and alfalfa crops in Qassim region

During a survey conducted in Qassim province, Saudi Arabia, in the year in 2015, 120 samples of carrot (Daucus carota subsp. sativus), onion (Allium cepa), faba bean (Vicia faba), green mustard (Brassica juncea) and alfalfa (Medicago sativa) plants displaying symptoms reminiscent of phytoplasma diseases were collected and tested for phytoplasma infection. Phytoplasma-specific PCR products were only amplified from symptomatic plants by nested-PCR. Disease incidence ranged from 3.14% in alfalfa crop fields 1 year after cultivation to 77.48% in 3-year-old fields. In the five carrot fields sampled in this study, the incidence changed from 3.2% to 100% after 7 months of cultivation. Phylogenetic analysis revealed that all Qassim phytoplasma isolates belong to the 16SrII group. Most of them shared 100% identity with papaya yellow crinkle (16SrII-D Y10097). The results from phylogenetic and virtual restriction fragment length polymorphism analyses of the 16S rRNA gene sequence confirmed that the phytoplasma of Qassim isolates under study is a member of 16SrII-D subgroup. To the best of my knowledge, the onion and green mustard are considered new hosts for the 16SrII group; therefore, this is the first report on the association of phytoplasma with diseases of faba bean, onion, carrot, mustard and alfalfa in Qassim province, Saudi Arabia.     

Detection and Identification of a 16SrII Group Phytoplasma Causing Clover Little Leaf Disease in Iran

White clover plants showing little leaf and leaf reddening symptoms were observed in Isfahan Province in central Iran. Restriction fragment length polymorphism analyses of nested PCR‐amplified fragments from Iranian clover little leaf phytoplasma isolates and representative phytoplasmas from other phytoplasma groups using AluI, CfoI, KpnI and RsaI restriction enzymes indicated that the clover phytoplasma isolates are related to the peanut WB group. Sequence analyses of partial 16S rRNA fragments showed that Iranian clover little leaf phytoplasma has 99% similarity with soybean witches'‐broom phytoplasma, a member of the peanut WB (16SrII) phytoplasma group. This is the first report of clover infection with a phytoplasma related to the 16SrII group.     

Novel insertion sequence-like elements in phytoplasma strains of the aster yellows group are putative new members of the IS3 family

Novel insertion sequence (IS)-like elements were isolated and characterized from phytoplasma strains in the aster yellows (AY) group (16SrI). The IS-like elements were cloned from phytoplasma strains AY1 and NJAY or PCR-amplified from 15 additional strains representing nine subgroups in the AY group using primers based on sequences of the putative transposases (Tpases). All IS-like elements contained sequences encoding similar Tpases of 321 amino acids (320 for strain CPh). Substantial amino acid sequence variability suggested multiple species of Tpases or IS-like elements exist in the AY phytoplasma group. These Tpases have an identical DDE motif that is most similar to the DDE consensus of Tpases in the IS3 family.     

Identification of phytoplasmas associated with sesame phyllody disease in southeastern Iran

Phyllody disease is a threat to sesame production in Kerman province, southeastern Iran. RFLP analysis of PCR products of phytoplasma-specific 16S rRNA gene (1.8 kb) and phylogenetic analyses of 16S-23S rDNA spacer region (SR) sequence indicated that the predominant agent associated with sesame phyllody in Kerman province is a phytoplasma with 100% similarity with eggplant big bud, and peanut witches’-broom phytoplasmas, members of “Candidatus Phytoplasma aurantifolia” from Iran and China, respectively. Among the samples tested, only one strain (SPhSr1), had a unique RFLP profile and its SR was 100% similar in nucleotide sequence with the phytoplasma carried by Orosius albicinctus and Helianthus annus witches’-broom phytoplasma from Iran, members of “Ca. Phytoplasma trifolii”. Virtual RFLP patterns of SPhJ2 (representative of the predominant PCR-RFLP profiles) SR sequence were identical to those of peanut witches’-broom phytoplasma (16SrII-A, JX871467). However, SPhSr1 SR sequence patterns resemble (99.7%) those of vinca virescence phytoplasma (16SrVI-A, AY500817).     

Cyclamen (Cyclamen persicum L.): a dead-end host species for 16Sr-IB and -IC subgroup phytoplasmas

Phytoplasmas belonging to the 16S rDNA subgroups IB and IC were found in five cyclamen (Cyclamen persicum L.) plants showing virescence and yellow stunted leaves and one plant showing phyllody, rolled and thickened leaves, respectively. Two cyclamens, representing the two syndromes, were chosen as source plants for transmission trials in which three leafhopper species, known as vectors of IB and IC subgroup phytoplasmas, were used to inoculate cyclamen and periwinkle [Catharanthus roseus (L.) G. Don] test plants. Out of 366 tested plants only one periwinkle exposed to Euscelis incisus was found harbouring a 16Sr‐IB phytoplasma. Out of 60 tested vector insects, only one adult of Macrosteles quadripunctulatus and two of E. incisus fed on 16Sr‐IB source cyclamen gave a positive amplification signal in nested PCR. This extremely low level of transmission to both cyclamen and the very susceptible periwinkle strongly suggests that cyclamen, commonly found infected in crops, is an unsuitable species for phytoplasma acquisition and can be regarded as a dead‐end host plant for phytoplasmas belonging to both IB and IC subgroups. Indications for glasshouse management are drawn from these findings. Among the leafhoppers investigated E. incisus falls most under suspicion since it feeds better than the others on cyclamen, was able to transmit the disease to one periwinkle plant, and IB phytoplasmas were detected in two individuals.     

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.     

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 Group 16SrVI Phytoplasma in Willows in China

In 2010 and 2011, willow proliferation disease was observed in Erdos, Inner Mongolia, China. The phytoplasma‐specific 16S rRNA gene fragment of 1.2 kb was amplified by a nested PCR with universal primer pair P1/P7 followed by R16F2n/R2. Phylogenetic and virtual RFLP analyses revealed that the phytoplasma associated with willow proliferation was a member of subgroup 16SrVI‐A. The field survey indicated that the incidence of willow proliferation in Erdos was approximately 36.84%. To our knowledge, this is the first record of group 16SrVI phytoplasma infecting willow in China.     

Detection of Putative Recombination Events in the 16S Ribosomal RNA Gene of Some Members of Candidatus phytoplasma

Recombination plays a major evolutionary role by creating genetic diversity and provides the potential to find rapid adaptation to new environmental conditions. We sought the occurrence of possible recombination events in the 16S ribosomal RNA gene of 60 accessions belonging to the group 16SrI of Candidatus phytoplasma (aster yellows phytoplasma). Three bioinformatic programs were used (TOPALI v2.5, RECCO and RDP package). All the three programs indicated the presence of putative recombination signals in aligned sequences. Recombination events located in the 16S ribosomal RNA gene revealed the presence of four recombining accessions gathering sugarcane grassy shoot phytoplasmas (JF928001, DQ459439, EF614269 and JN223446).     

Lethal yellowing-type diseases of palms associated with phytoplasmas newly identified in Florida, USA

A nested polymerase chain reaction (PCR) assay was used to detect phytoplasmas in stem tissues from declining silver date ( Phoenix sylvestris ), Canary Island date ( Phoenix canariensis ), edible date ( Phoenix dactylifera ), Queen ( Syagrus romanozoffiana ) and Mexican fan ( Washingtonia robusta ) palms, all displaying symptoms similar to lethal yellowing (LY) disease, in a tri-county region of west central Florida. Restriction fragment length polymorphism analysis or sequencing of PCR-amplified rDNA products (1.6 kb) identified three distinct group 16SrIV phytoplasma strains among 38 infected palms. Most palms (86.8%) contained Texas Phoenix decline (TPD) phytoplasma, a subgroup 16SrIV-D strain, while two P. canariensis harboured palm LY phytoplasma, a subgroup 16SrIV-A strain. A novel phytoplasma, initially detected in W. robusta and tentatively classified as a subgroup 16SrIV-F strain, also occurred with LY phytoplasma as mixed infections in two P. dactylifera palms. Collectively, these findings extend the known geographic range of TPD in the USA to include Florida and the listing of TPD-susceptible palm species to include P. dactylifera , P. sylvestris and S. romanozoffiana . Moreover, discovery of a novel subgroup 16SrIV-F strain in W. robusta adds to the list of phytoplasma host palm species and complexity of phytoplasma-vector-palm pathosystems newly identified in the west central region of the state. The emergence of new lethal disease of palms beyond southern subtropical region of the state is unprecedented but follows similar developments in other regions where LY is known to occur.     

Detection and variability of the lethal yellowing group (16Sr IV) phytoplasmas in the Cedusa sp. (Hemiptera: Auchenorrhyncha: Derbidae) in Jamaica

Lethal yellowing (LY) group phytoplasma was detected in members of the Cedusa species of Derbids from Jamaica by nested PCR assay employing the rRNA primer pairs P1/P7 and LY16Sf/LY16Sr. A 1400‐bp product was obtained from 13/43 Derbids that were analysed. Restriction fragment length polymorphism analysis of the nested PCR product revealed variations in 6/13 Derbids analysed, suggesting that the planthoppers could be infected with different strains of the LY group phytoplasma. Sequence analysis of the 16S rDNA gene determined that the percentage of similarity of the phytoplasma strains in the Derbids ranged from 97–98% to the phytoplasma strain found in coconuts in Florida (LYFL‐C2) and Jamaica (LYJ‐C8). The phytoplasma strains in the Derbids clustered together with the Jamaican and Florida coconut LY phytoplasma, as well as other characterised strains composing the LY phytoplasma (16Sr IV) group.     

Characterization of Phytoplasmas Related to 'Candidatus Phytoplasma asteris' and Peanut WB Group Associated With Sweet Cherry Diseases in Iran

Symptoms suggestive of phytoplasma diseases were observed in infected sweet cherry trees growing in the central regions of Iran. Phytoplasmas were detected in symptomatic trees by the nested polymerase chain reaction (nested PCR) using phytoplasma universal primer pairs (P1/Tint, PA2F/R, R16F2/R2 and NPA2F/R). Restriction fragment length polymorphism analyses of 485 bp DNA fragments amplified in nested PCR revealed that different phytoplamas were associated with infected trees. Sequence analyses of phytoplasma 16S rRNA gene and 16S-23S intergenic spacer region indicated that the phytoplasmas related to ' Ca. Phytoplasma asteris ' and peanut WB group infect sweet cherry trees in these regions. This is the first report of the presence of phytoplasmas related to ' Ca. Phytoplasma asteris' and peanut WB group in sweet cherry trees.     

Identification of two new phylogenetically distant phytoplasmas from Senna surattensis plants exhibiting stem fasciation and shoot proliferation symptoms

Sunshine trees (Senna surattensis) exhibiting unusual stem fasciation symptoms were observed in Yunnan, China. Morphological abnormalities of the affected plants included enlargement and flattening of stems and excessive proliferation of shoots. An electron microscopic investigation revealed presence of single membrane bound mycoplasma‐like bodies in sieve elements of symptomatic plants. With DNA templates extracted from diseased plants and phytoplasma universal primers P1/P7 and P1A/R16S‐SR, nested polymerase chain reactions produced amplicons of 1.5 kb. Subsequent restriction fragment polymorphism and nucleotide sequence analyses of the amplicons indicated that the diseased plants were infected by distinct phytoplasmas affiliated with two phylogenetically distant taxa classified in two 16Sr groups (16SrXII and 16SrV). This is the first report that sunshine tree is a natural host of two evolutionarily divergent phytoplasmas and the first report that a ‘Candidatus Phytoplasma australiense’‐related strain is present in China. The findings signal a significant expansion of both geographical distribution and host range of 16SrXII and 16SrV phytoplasmas.     

Molecular identification of phytoplasmas in fasciated cacti and succulent species and associated hormonal perturbation

Fasciation, a frequent phenomenon in Cactaceae, has been attributed to various causes. The present study reports on phytoplasma-induced fasciation in Euphorbia coerulescens (Euphorbiaceae), Orbea gigantea (Asclepiadaceae), Opuntia cylindrica (Cactaceae), and Senecio stapeliiformis (Asteraceae). DNA was extracted from symptomless and fasciated tissues and amplified by nested PCR using universal primers P1/P7 followed by R16F2n/R16R2 produced amplicons of 1.2 Kb. The nucleotide sequence analyses of the amplicons indicated that fasciated plants were infected by phytoplasma. Phylogenetic analysis placed the cacti fasciation phytoplasmas in 16SrII group. The hormonal content of symptomless and fasciated tissues including indole-3-acetic acid (IAA), kinetin (Kin), N⁶-benzyladenine (BA), abscisic acid (ABA), and gibberellic acid (GA₃) was quantified by high performance liquid chromatography (HPLC). The results indicated that fasciation in O. gigantea was correlated with the accumulation of Kin and IAA increasing five and two times, respectively, as compared to symptomless tissue. However, there was no consistent pattern of hormones in other fasciated species (E. coerulescens, O. cylindrica, and S. stapeliiformis), suggesting that different plant species might have different mechanism to develop fasciation associated with phytoplasma infection.     

Effect of indole-3-butyric acid on phytoplasmas in infected Catharanthus roseus shoots grown in vitro

Phytoplasmas are noncultivable bacteria usually maintained in Catharanthus roseus shoots grown in vitro on MS medium with benzylaminopurine. The aim of our research was to examine the influence of indole-3-butyric acid (IBA) on C. roseus shoots infected with three different phytoplasma strains. Supplement of IBA in the medium supported plant growth, photosynthesis and remission of symptoms in all phytoplasma-infected shoots, but had no effect on the presence of EY-C and SA-I phytoplasma strains in tested tissue. However, HYDB phytoplasma was undetectable in approximately half of the tested shoots grown on the medium with IBA. After 1 year of IBA treatment, HYDB-infected periwinkle shoots were retransferred to the medium supplemented with benzylaminopurine. Some of the shoots showing remission of symptoms during the IBA treatment permanently escaped the infection and remained negative when tested for phytoplasma presence. This is the first report on the differential influence of plant growth regulators on phytoplasma-infected C. roseus shoots.     

Molecular detection and identification of phytoplasmas associated with Catharanthus roseus,Pinus eldarica,and Petunia hybrida in southeastern Iran's urban green spaces

Given the potential for urban green spaces to provide fresh and healthy environments for humans, exploring the issues that threaten plants in these places is crucial. Phytoplasma-related symptoms were encountered on some plants in urban green spaces in the province of Kerman, southeastern Iran, between 2017 and 2019. Affected periwinkles and petunias exhibited phytoplasma disease symptoms, including virescence, phyllody, and witches'-broom. However, ball or disc-like shoot proliferation symptoms were noticed on the trunks and branches of pine trees. PCR was performed with phytoplasma-detecting universal primers, targetting and amplifying the 16S rRNA gene, and determining whether phytoplasmas are implicated in the symptomatic plants. The infection of the symptomatic plants was confirmed using nested-PCR amplification of expected DNA sizes for phytoplasmas. No product, however, was amplified from sampled symptomless plants. The sequencing of nested-PCR products was performed to obtain sequences encasing the standard F2nR2 fragments. The resulted sequences were submitted to iPhyClassifier, the universal phytoplasma classification platform, for the taxonomic assignment of the found phytoplasmas compared with previously identified ‘Candidatus Phytoplasma’ species, groups, and subgroups. The results revealed that phytoplasma strains related to the species ‘Ca. P. trifolii’ (16SrVI-A subgroup) infect periwinkles and pines. However, strains from the species ‘Ca. P. aurantifolia’ (16SrII-D subgroup) and ‘Ca. P. phoenicium’ (16SrIX-C subgroup) were found in petunias and periwinkles, respectively. To the best of our knowledge, phytoplasmas from the 16SrVI-A and 16SrII-D subgroups are the first reported to infect these plants in Kerman province, while a related strain from the subgroup 16SrIX-C is the first recorded to infect periwinkles in Iran and the second in the world.     

Detection of a 16SrXII phytoplasma associated with sugarcane yellow leaf syndrome in India

Phytoplasma strain was detected in leaves of sugarcane in India exhibiting symptoms of yellowing of midribs. A phytoplasma characteristic 1.2 kb rDNA PCR product was amplified from DNAs of all diseased samples but not in healthy sugarcane plants tested using phytoplasma universal primer pairs P1/P7 and f5U/r3U. Restriction fragment length polymorphism (RFLP) analysis of amplified 16S rDNA indicated that diseased sugarcane was infected by phytoplasma. The 16S rDNA sequence of the Indian sugarcane yellow leaf phytoplasma (SCYLP) showed the closest identity (99%) to that of SCYLP in Cuba identified as Macroptilium lathyroides (AY725233), which belongs to 16SrXII (Stolbur group). This is the first record of the detection of SCYLP, and identification of the 16SrXII group of phytoplasma associated with yellow leaf syndrome (YLS) in India.     

Identification,occurrence, incidence and transmission of phytoplasma associated with Petunia violacea witches’ broom in Iran

A petunia witches’ broom (PvWB) disease, characterized by phyllody, virescence, witches’ broom, little leaf and yellowing, was observed in municipal lands and parks in Bandar Abbas, Hormozgan province, Iran. The disease was present with an average incidence of 20%. PCR and sequencing analysis carried out on selected samples from symptomatic plants showed the presence of a phytoplasma associated with the disease. The molecular comparison of the 16S ribosomal gene indicated 99% sequence identity with the one of “Candidatus Phytoplasma australasia”. This phytoplasma was transmitted to healthy petunia plants under experimental conditions by the leafhopper Orosius albicinctus that was then demonstrated to be a vector of this phytoplasma.     

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).     

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.