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.     

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.     

寡核苷酸管芯片技术检测和鉴别我国不同组植原体

[目的]不同组植原体检测和鉴别的特异性探针已有报道,为了筛选出适合于我国不同组植原体检测和鉴别的特异性探针,建立管芯片检测和鉴别植原体技术,并对我国发生的疑似植原体病害进行鉴别。[方法]通过PCR扩增结合管芯片杂交技术,对收集到的15种植原体侵染的植物样品及其健康对照进行检测和鉴别。[结果]建立了管芯片检测和鉴别植原体技术体系。15种病害样品中,13种获得显著的阳性杂交信号,并且所有的健康对照都呈现为阴性。13种植原体病害依16Sr DNA直接测序可分为16SrⅠ、Ⅱ、Ⅴ、XIX四组植原体。在所有探针中,植原体的通用探针(Pp-502)可以检测到所有确定的植原体样品。16SrⅠ组特异性探针(PpⅠ-465)可以确定16SrⅠ组的泡桐丛枝、苦楝丛枝、桑树萎缩和莴苣黄化4种植原体样品。16Sr II组特异性探针(PpⅡ-629)仅可以确定16Sr II组的花生丛枝、甘薯丛枝和臭矢菜丛枝3种植原体样品。但16Sr V组的枣疯病、樱桃致死黄化和重阳木丛枝及16Sr XIX组的板栗黄化皱缩植原体与其他组专化性探针皆有明显的交叉杂交信号。相比于PCR扩增的凝胶电泳检测,管芯片检测的灵敏度提高了1 000倍。对疑似植原体病害的诊断结果显示河南濮阳的红花槐丛枝的病原应为16Sr V组植原体,福建福州的长春花黄化丛枝应为16SrⅠ组植原体;而北京戒台寺牡丹黄化皱叶和内蒙古包头柳树丛枝未出现任何植原体专化的杂交信号。[结论]管芯片杂交技术作为一种检测和鉴别植原体的方法,可应用于我国植原体病害调查和诊断,并为植原体的鉴别和分类提供可靠的依据。     

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.     

“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.”     

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.     

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.     

早竹丛枝病的调查及病原菌的分子鉴定

[目的]对早竹丛枝病进行调查及病原菌分子鉴定,为早竹丛枝病的病害诊断和防治提供科学依据。[方法]采用单株水平和单枝盘水平的2种病害分级标准对早竹丛枝病进行调查。使用植原体16S rDNA和真菌rDNA-ITS序列的特异性PCR引物,对早竹丛枝病的病原菌进行分子鉴定。[结果]调查的6块样地早竹丛枝病的平均发病率为18.59%,平均病情指数为6.67。感病的早竹DNA样品能够扩增出真菌的rDNA-ITS序列,而不能够扩增出植原体的16S rDNA序列;扩增出的序列与报道的竹针孢座囊菌的序列同源性达到99.00%,与其它真菌的序列同源性最高仅为94.00%。[结论]浙江省德清县早竹丛枝病的病原菌为竹针孢座囊菌。     

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.     

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 .     

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.     

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.     

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.     

Identification of ‘Candidatus phytoplasma ziziphi’ associated with persimmon (Diospyros kaki Thunb.) fasciation in China

Persimmon (Diospyros kaki Thunb.) trees, with fasciation symptoms (PF), were observed in an orchard located in suburban Tai'an, Shandong Province, China. A specific fragment of the phytoplasma 16S rRNA gene, approximately 1.2 kb in length, was amplified from two symptomatic plants via nested polymerase chain reaction, while no fragment was obtained from healthy controls. The two samples (PF1 and PF2) resulted with 99.5% nucleotide sequence identity. Phylogenetic and restriction fragment length polymorphism (RFLP) analysis of 16S rRNA gene sequences revealed that PF1 was a member of ribosomal subgroup B of the elm yellows group (16SrV), and PF2 may represent a novel subgroup within the 16SrV group, designed as 16SrV‐I. This is the first report of phytoplasmas of the 16SrV group associated with persimmon fasciation disease.     

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 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.     

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.     

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.     

从我国20种感病植物中扩增植原体16S rDNA片段及其RFLP分析

本研究收集了２０种感染植原体的植物材料,从患病材料和相应健康植物组织中提取总ＤＮＡ用作ＰＣＲ模板,选择两对通用引物进行巢式ＰＣＲ,扩增植原体的１６ＳｒＤＮＡ片段,回收扩增产物,通过限制性酶切片段长度多态性（ＲＦＬＰ）分析进行分类。２０种患病植物材料中的植原体有１３种属于翠菊化种,２种属于白对黄化种,３种属于花生丛枝种,２种属于三叶草丛生种。     

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.