Molecular characterization of a phytoplasma associated with Euonymus bungeanus witches’ broom in China

Euonymus bungeanus plants exhibiting symptoms of abnormal branches, small leaves and phyllody, which is indicative of E. bungeanus witches’ broom (EbWB) disease, have recently been found in Beijing, China. A phytoplasma from symptomatic E. bungeanus plants was identified by 16S rRNA polymerase chain reaction (PCR) using the phytoplasma‐specific universal primer pair R16mF2/R16mR1. Inoculation of healthy E. bungeanus plants by grafting with diseased scions was also performed. The rp and secY genes of the EbWB phytoplasma were cloned and sequenced as was the 16S rRNA gene. Sequence and phylogenetic analyses of 16S rRNA, rp and secY genes indicated that the phytoplasma associated with E. bungeanus belongs to the 16SrV‐B, rpV‐C and secY‐C subgroup, the same subgroup as the jujube witches’ broom (JWB) phytoplasma that is widely distributed among jujube trees in China. Comparative analyses based on virtual restriction fragment length polymorphism (RFLP) showed that the EbWB phytoplasma is more closely related to another 16SrV‐B subgroup strain: RPWB (Robinia pseudoacacia witches’ broom). To the best of our knowledge, this is the first report of a witches’ broom phytoplasma in E. bungeanus in China, and the findings add a new cultivated plant species to the already broad natural host range of 16SrV‐B subgroup phytoplasmas.     

Identification of a phytoplasma associated with Syringa reticulata witches' broom disease in China

During the summer of 2018, one Syringa reticulata plant showing witches' broom and small leaves was observed in Beijing, China. Molecular diagnostic tools and electron microscopic cell observation were used to detect the possible pathogen of this disease. As a result, the phytoplasma in the symptomatic S. reticulata tree was confirmed by amplifying the 16S rRNA gene using the phytoplasma‐specific universal primer pair R16mF2/R16mR1 and observation with transmission electron microscopy. The rp and tuf genes of the phytoplasma were also cloned and sequenced as the 16S rRNA gene. Sequence and phylogenetic analyses of the 16S rRNA, rp and tuf genes indicated that the phytoplasma associated with S. reticulata witches' broom (SrWB) disease belonged to the 16SrV‐B subgroup, and it was closely related to the 16SrV‐B subgroup phytoplasma strain jujube witches' broom, which causes serious disease of jujube fruit trees in China. This study shows the S. reticulata tree as a new host of a phytoplasma belonging to the 16SrV‐B subgroup in China.     

Identification and molecular characterization of 16SrV group phytoplasmas with flowering cherry in China

Flowering cherry (Prunus serrulata) trees showing symptoms of excessive axillary growth, stunting, short internodes and decline were observed in Yantai City, Shandong province, China. The samples with the flowering cherry decline were designated as FcD‐YT phytoplasma. Sequencing results, restriction fragment length polymorphism (RFLP) and phylogenetic analysis of sequences from the 1248‐bp R16F2n?R16R2 products and partial ribosomal protein operon genes revealed the FcD‐YT phytoplasma as a member of subgroup 16SrV‐B, rpV‐C. Two RFLP patterns were observed in recombinant colonies from the cloned 16S rDNA gene with endonucleases HpaII and MseI. To our knowledge, this is the first report of 16SrV group phytoplasma declining flowering cherry trees in China.     

Molecular identification of a phytoplasma associated with Elm witches’‐broom in China

Elm samples with and without witches’‐broom symptoms (EWB) were collected from Tai’an and Zhaoyuan, Shandong Province, China. Phytoplasmal cells were observed in the phloem cells of symptomatic plants under electron microscope. Specific fragments of about 1.2 kb in length were amplified with nested‐PCR from symptomatic samples, while no fragment was obtained from healthy plants. The 16S rRNA gene sequences of the phytoplasmas associated with elm witches’‐broom in Tai’an (EWB‐TA) and Zhaoyuan (EWB‐ZHY) had high similarities, and formed a sublineage in phylogenetic tree, with members of subgroup B or D of aster yellows group (16SrI). Computer simulated restriction fragment length polymorphism analysis of 16S rRNA gene revealed that EWB‐TA and EWB‐ZHY patterns had similarity coefficients of 1.00 with the pattern from the representative strains of subgroup 16SrI‐B, and had a similarity coefficient of lower than 0.97 with representatives of other subgroups. These results indicated that the phytoplasma strain associated with elm witches’‐broom in China was very closely related to ‘Candidatus Phytoplasma asteris’ OAY, belonging to subgroup‐B of aster yellows group (16SrI‐B). This is the first report of a phytoplasma associated with elm witches’‐broom disease in China.     

Phytoplasma associated with Chinese tallow tree yellowing disease in China represents a new 16SrIII subgroup

Recently, samples of the Chinese tallow tree (Sapium sebiferum) displaying yellowing symptoms were collected from a grove in Tai'an, Shandong Province, China. The association of phytoplasma with yellowing disease was ascertained using nested polymerase chain reaction (PCR) of the 16S rRNA gene by using the phytoplasma‐specific universal primer pair P1/P7, followed by R16F2n/R16R2 as nested primers, and rp genes primed using rpL2F3/rp(I)R1A followed by rp(III)‐FN/rp(I)R1A. The sequence and phylogenetic analyses of the 16S rRNA gene and rp genes revealed that the phytoplasma associated with the Chinese tallow tree belonged to the 16SrIII group (the X‐disease group). Computer‐simulated and gel‐based restriction fragment length polymorphism (RFLP) analyses revealed that the RFLP patterns were different from the reference patterns of all previously established 16SrIII subgroups, with the maximum similarity coefficient exceeding the threshold for delineation of a new subgroup RFLP pattern type within the 16SrIII group. Thus, the phytoplasma associated with the Chinese tallow tree yellowing disease, designated as ‘CTTY’, represents a new subgroup (16SrIII‐Y). This study shows the Chinese tallow tree as a new host of phytoplasma belonging to the 16SrIII group in China and worldwide.     

Molecular characterization of a phytoplasma from the aster yellows (16SrI) group naturally infecting Populus nigra L. ‘Italica’ trees in Croatia

Leaf and branch samples were collected from 10 Populus nigra L. ‘Italica’ trees found in the Zagreb urban area. One of the P. nigra L. ‘Italica’ trees exhibited leaf yellowing, overall sparse foliage, stunting and decline. Two methods for the nucleic acid extraction in the phytoplasma detection from P. nigra were compared. A phytoplasma from the aster yellows group (16SrI) was detected by PCR in the symptomatic as well as in four apparently asymptomatic plants. The pathogens are classified, by restriction fragment length polymorphism (RFLP) analysis of the 16S rRNA gene plus the spacer region, as members of a newly described subgroup 16SrI‐P. Phylogenetic analysis of 16S ribosomal and spacer region sequence confirmed their close relationship with the other members of the aster yellows group. However, RFLP analyses of other conserved genes such as tuf, BB88 and ribosomal protein rpL22 gene, clearly confirmed that this is a molecularly distinguishable phytoplasma belonging to a new ribosomal protein subgroup designated rp‐O.     

Salix tetradenia Hand.‐Mazz: a new natural plant host of ‘Candidatus phytoplasma’

This article reports Salix tetradenia Hand.‐Mazz as a new host of Candidatus phytoplasma and demonstrates its association with witches' broom disease on S. tetradenia plants. Plants exhibited typical visual symptoms of phytoplasma with virescence, abnormality of flowers and witches' broom, and phytoplasma bodies were observed by transmission electron microscopy. Products of 1.2 kb were amplified by nested PCR using phytoplasma universal primer pairs R16F2n/R16R2, but no amplification products were obtained from symptomless plants. The sequence analysis of three 16S rDNA isolates showed 99.84%, 99.68% and 99.76% identify, respectively, with the homologous gene (nc_005303) of member of ‘Candidatus phytoplasma asteris’ (16SrI) group. Phylogenetic and virtual computer‐simulated restriction fragment length polymorphism analysis of the 16S rRNA, tuf and rp gene sequences confirmed that this phytoplasma clustered in the 16SrI‐B subgroup. These results indicated that the diseased S. tetradenia plants were infected by a phytoplasma of the 16SrI group. This is the first report on the occurrence of phytoplasma disease on S. tetradenia worldwide.     

Molecular identification of a phytoplasma associated with Sophora Root yellows

A phytoplasma infecting Sophora Root (Sophora alopecuroides) was detected and identified in Alar, Xinjiang Uygur Autonomous Region of China. Typical phytoplasma bodies were observed in sieve tubes of the diseased plants by transmission electron microscopy. A partial 16S rRNA gene and ribosomal protein (rp) genes containing rpl22 (rplV) and rps3 (rpsC) were amplified by direct and nested PCR. Based on the sequence similarity of the 16S rRNA and rp genes with accompanying phylogenetic analyses, the phytoplasma associated with Sophora Root yellows belongs to the 16SrI group (aster yellows group). Virtual RFLP analysis of these 16S rRNA and rp gene sequences showed distinct differences from those of reference phytoplasma strains representing previously described subgroups of the 16SrI group. Moreover, the similarity coefficient (0.92) of the RFLP profile of this phytoplasma was less than the threshold similarity coefficient (0.97) required for subgroup classification. Thus, the phytoplasma isolate of Sophora Root plants, designated as ‘SoRY’, represents a new subgroup. Furthermore, this is the first report of phytoplasma disease associated with Sophora Root plants.     

A new phytoplasma associated with witches’‐broom on Japanese maple in China

In September 2011, five Japanese maple (Acer palmatum Thunb.) trees with symptoms of witches’‐broom were observed growing near each other at a maple grove in Northwest A&F University, Yangling, Shaanxi Province, China. Pleomorphic phytoplasma‐like bodies were observed in the phloem sieve tube elements of symptomatic plants under transmission electron microscope (TEM). The presence of phytoplasma was further confirmed by a nested polymerase chain reaction (PCR), which amplified a 1.2‐kb fragment using universal primer pair R16mF2/R16mR1 followed by further amplification using primer pair R16F2n/R16R2. Phylogenetic analysis and gel‐based restriction fragment length polymorphism (RFLP) analysis demonstrated that the Japanese maple witches’‐broom was associated with phytoplasma belonging to subgroup 16SrI‐D. This is the first report of a phytoplasma disease of Japanese maple.     

Genetic variability of Alder yellows phytoplasma in Alnus glutinosa in its natural Spreewald habitat

The genus ‘Candidatus Phytoplasma’ comprises wall‐less bacteria colonizing the phloem of plants and insect tissues. Phytoplasmas are associated with diseases in over 1000 plant species worldwide, including many important crops as well as forest trees. Alder yellows (AldY) phytoplasma, which frequently infects Alnus spp., is closely related to the economically important phytoplasma causing Flavescence dorée (FD) in grapevines. In a natural habitat (Spreewald, Brandenburg, Germany), 57 Alnus glutinosa (black alder) trees were examined for phytoplasma infection in summer 2013. No phytoplasma typical infection‐associated symptoms such as yellowing and decline were observable in this natural swamp‐alder area. Amplification followed by a restriction fragment length polymorphism, and a sequence analysis of the 16S rDNA, allowed for the detection of AldY phytoplasmas in all examined trees and their assignment to the taxonomic group 16SrV‐C. Additional analyses of the non‐ribosomal marker gene methionine aminopeptidase (map) revealed diverse strains as well as mixed infections with closely related AldY strains, and the strains were assigned to phylogenetic clusters closely related to German Palatinate grapevine yellows, AldY or FD strains. The results confirmed that AldY phytoplasmas infection in A. glutinosa is prevalent. The results also indicate a presence of an established phytoplasma population in chronically infected black alder.     

Elm yellows: A phytoplasma disease of concern in forest and landscape ecosystems

Elm yellows (EY) is a lethal or decline phytoplasma disease that affects several Ulmus (elm) species and hybrids, which is widespread in North America and Europe. The symptoms vary among the elm species. In those native to North America, main symptoms include epinasty, chlorosis, premature casting of the leaves, yellow to brown discoloration of the phloem in the roots and stem and tree death that usually occurs within 1 or 2 years from the appearance of foliar symptoms. In contrast, affected trees of European and Asian species are primarily characterized by witches’ broom as a specific symptom, do not show phloem discoloration and are less prone to decline. The disease is caused by a relatively genetically homogeneous phytoplasma, the EY agent “Candidatus Phytoplasma ulmi,” a member of the EY phytoplasma group or 16SrV group, subgroup 16SrV‐A. In nature, this pathogen exhibits a high plant host specificity. The elm leafhopper Scaphoideus luteolus is the only confirmed vector of EY phytoplasma in North America, whereas Macropsis mendax has been reported as a natural vector in Northern Italy. However, other insect vectors are likely to be involved in its natural spread. Phytoplasmas of other taxonomic groups or 16SrV subgroups, which are known to infect a wide range of plant hosts, have been identified in naturally infected elm trees. However, the pathological relevance of these “non‐elm” phytoplasmas needs to be confirmed in many cases. Their detection is mainly based on the highly sensitive nested PCR assays, while pathological data are lacking. This study summarizes, within the framework of a single comprehensive review, the current knowledge of EY. Gaps in knowledge of this disease and prospects for future research are also critically discussed.     

Rapid detection and identification of ‘Candidatus Phytoplasma pini’‐related strains based on genomic markers present in 16S rRNA and tuf genes

In order to devise a method for rapid detection of ‘Candidatus (Ca.) Phytoplasma pini’ and for distinguishing it rapidly from other phytoplasmas, we carried out preliminary sequencing of Lithuanian ‘Ca. Phytoplasma pini’ strain PineBL2 using Illumina (NGS) technology and targeted sequencing employing universal phytoplasma primers. We focused on two resulting chromosomal segments that contained a 16S rRNA gene and a translation elongation factor EF‐TU gene (tuf), respectively. Based on alignments of the ‘Ca. Phytoplasma pini’ gene sequences with the corresponding sequences of other phytoplasmas, we designed new primer pairs for PCR‐based detection of ‘Ca. Phytoplasma pini’. Because ‘Ca. Phytoplasma pini’ strains are expected to reside in the pine phloem in a very low titre, one might expect that they could be detected only by nested PCR. By contrast, the primers and PCR protocols designed in the current work enabled rapid direct PCR detection and identification of ‘Ca. Phytoplasma pini ’ by amplifying a 484 bp 16S rDNA segment and a 513 bp tuf gene fragment that contain regions unique to this phytoplasma .     

Genetic diversity of the hyperparasite Sphaerellopsis filum on Melampsora willow rusts

The non‐specific rust hyperparasite Sphaerellopsis filum occurs naturally on Melampsora rusts of many species of the genus Salix as well as on a large range of other rust genera worldwide. To study the genetic diversity of the hyperparasitic fungus 77 S. filum isolates collected from rusts on willow clones from plantations, clone collections and natural habitats of different sites were investigated using polymerase chain reaction ‐ restriction fragment length polymorphism (PCR‐RFLP) analysis of the rDNA internal transcribed spacer regions including 5.8S rDNA and sequence analysis. Additionally, strains from Melampsora poplar rusts (4) and strains of Puccinia abrupta from Parthenium hysterophorus (5) and of P. obscura from Bellis perennis (1) were used for comparisons. Results of genetic analysis demonstrated distinct variation within the S. filum isolates. Two main groups with more than 32% difference between their nucleotide sequences were distinguished, indicating two taxa within S. filum. Within the first main group three profiles (I, II, III) were detected. The differences between these profiles were about 12%. The variation within each profile was very low (less than 2%). The second main group comprised two profiles (IV, V), which differed in 12 to 16% of their nucleotide positions. The isolates of group IV possessed a higher variation (up to 5%) within the group than those of the first main group (I, II, III). Group V was only represented by a single isolate. Neither interrelations between the S. filum profiles and the Melampsora genotypes nor a spatial distribution could be detected. It is remarkable that the six strains of S. filum from Puccinia rusts belong to one subgroup.     

“Candidatus Phytoplasma solani” associated with Eucalyptus witches’ broom in Iran

During summer of 2015, Eucalyptus camaldulensis plants showing witches’ broom, little leaf and general yellowing of the foliage were observed in west of Fars and Khozestan province of Iran. DNA from samples of 22 symptomatic and two asymptomatic trees was extracted and subjected to molecular analyses. Nested‐PCR test using R16F2n/R16R2 primers confirmed phytoplasma presence in 63% of symptomatic Eucalyptus plants. Sequence analysis along with virtual RFLP of the 16S ribosomal DNA allowed to classify three Eucalyptus witches’ broom strains into the “stolbur” (“Candidatus phytoplasma solani”) 16SrXII‐A subgroup. Comparison of the secA and secY gene sequences with sequences deposited in GenBank confirmed the phytoplasma identity. Real and virtual RFLPs of the amplified secY gene using HaeIII, MseI and RsaI restriction enzymes showed profiles indistinguishable from each other. This is the first study reporting E. camaldulensis as a new host species for “Ca. P. solani.”     

Campsisgrandiflora as a new host species harbouring two novel 16SrI subgroups of phytoplasmas

In September 2019, two diseased plants of Campsis grandiflora showing the main symptom of witches' ‐broom (CgWB) were found in a nursery garden in Yangling, Shaanxi province, China. Partial 16S ribosomal RNA (F2nR2 region) and ribosomal protein (rp) genes of phytoplasmas were generated from the symptomatic plants by PCR amplification, and phytoplasma bodies were observed in the sieve tube elements of the CgWB samples under a transmission electron microscope, indicating phytoplasma infection in the two CgWB plants. Restriction fragment length polymorphism analysis of the F2nR2 region and similarity coefficient results suggested that the two associated phytoplasmas belong to two novel subgroups of 16SrI (aster yellows) group, designated as AK and AL. On the reconstructed phylogenetic trees based on F2nR2 regions and rp genes of phytoplasmas, respectively, the CgWB‐associated phytoplasmas clustered together with members of 16SrI subgroups. This was the first record of phytoplasmas infecting C. grandiflora worldwide.     

First report of 16Sr II‐C subgroup phytoplasma association with Acacia mangium in Tripura,India

Leaf yellowing symptoms were observed on Acacia mangium in the Sipahijala district of Tripura, India, during June 2017. Symptomatic and asymptomatic leaf samples (three of each) were collected from roadside trees of A. mangium for DNA extraction using the CTAB method. Amplicons of ~1.25 kb and ~480 bp were detected in all the symptomatic samples using the phytoplasma‐specific universal 16S rRNA and secA gene primers. Pair wise sequence analysis of 16S rRNA gene sequences, virtual RFLP and phylogenetic analysis revealed that the phytoplasma strain associated with A. mangium belonged to phytoplasma subgroup 16SrII‐C. This is the first report of an association between the 16SrII‐C subgroup and A. mangium leaf yellowing.     

A comparison on DNA methylation levels in bamboo at five developmental stages

Bamboo shoots grow quickly through the rapid elongation of internodes. In order to verify whether DNA methylation affects bamboo shoot elongation, we compared DNA methylation levels at five developmental stages of bamboo (Phyllostachys praecox), including young bamboo shoot of 40-cm at length (S1), bamboo shoot of 2-cm at length (S2), young leaf (Y1), leaf (Y2), and aging leaf (M), by using amplified fragment length polymorphism (AFLP) based on KpnI/MseI and Acc65I/MseI platforms. The polymorphism ratio of methylated to unmethylated DNA sites in bamboo shoot (S1 and S2) was higher than that in leaf (Y1, Y2 and M). The polymorphism ratio of methylated to unmethylated DNA sites in S2 was highest (32%). Our results suggest that DNA methylation changed greatly at bamboo growing stages, especially in stage of rapid elongation of internodes.     

Detection of phytoplasma infections in declining Populus nigra ‘Italica’ trees and molecular differentiation of the aster yellows phytoplasmas identified in various Populus species

A disease of Populus nigra‘Italica’ associated with foliar yellowing, sparse foliage, stunting, dieback, and decline was observed in south-western Germany; a witches’ broom disease of Populus alba that is known in other countries was also detected in Hungary and Germany. The aetiology of the diseases was studied by fluorescence microscopy and polymerase chain reaction (PCR) amplification. Using fluorescence microscopy, phytoplasmas could be detected only in P. alba. However, most diseased trees of P. nigra‘Italica’ tested phytoplasma-positive by PCR. In some of the trees the phytoplasma numbers were so low that nested PCR was required to detect the infection. Very low phytoplasma numbers were also observed in diseased Populus tremula. The identity of phytoplasmas from P. nigra‘Italica’ sampled in Germany and France, P. alba and also P. tremula was examined by restriction fragment length polymorphism (RFLP) analysis of PCR-amplified ribosomal DNA. In all poplars, phytoplasmas of the aster yellows group were detected. However, three different RFLP groups were identified that consisted of (1) French strains from P. nigra‘Italica’, (2) German strains from P. nigra‘Italica’ and (3) strains from P. alba and P. tremula. The profile observed in the last group was probably the result of sequence heterogeneity in the two 16S RNA genes.     

Molecular identification and characterization of a new phytoplasma strain associated with Chinese chestnut yellow crinkle disease in China

Chinese chestnut trees (Castanea mollissima BL.) planted in a suburb of Beijing, China developed symptoms including yellowing, leaf crinkling, little leaf, shortened internodes, and empty burrs. Transmission electron microscopy revealed presence of phytoplasma cells in phloem sieve elements of the symptomatic chestnut trees. Molecular cloning and sequence analysis of PCR‐amplified near‐full length 16S rRNA gene indicated that the phytoplasma associated with the Chinese chestnut yellow crinkle disease is closely related to Japanese chestnut witches’‐broom phytoplasma. This is the first report of a phytoplasmal disease in Chinese chestnut trees.     

木麻黄青枯病菌的分离及强致病菌株的筛选

[目的]筛选出强致病菌株用于木麻黄抗病育种研究工作。[方法]在广东沿海木麻黄青枯病发病区采集病根,开展病原菌两种不同分离方法的比较研究,对分离出的31个病株进行16s rRNA测序鉴定及致病性测定。[结果]采用稀释分离法及根系溢出法在TTC培养基上共分离出了31个病原菌株,根系溢出法操作简便,杂菌含量低,分离率在60%左右,可作为常规稀释分离法的补充。31个菌株进行分子鉴定,只有22个菌株扩增出了特异性条带,经测序比对确定这22个菌株为青枯菌。青枯菌株致病性测定结果显示菌株致病性在无性系间、菌株间及菌株与无性系间的交互作用均具有极显著差异(P0.01),不同接种方法间菌株致病性相关系数值较小,介于0.496 6~0.731 0之间,即室内水培接种与小苗盆栽接种不存在密切的直线相关关系。[结论]综合选择在不同无性系及不同接种方法中均具有较强致病性的GL-2、H、M、TC-1、F、Q菌株作为下一步木麻黄种质资源抗性鉴定及抗病育种研究试验菌株。