‘Candidatus Phytoplasma ziziphi’ associated with Sophora japonica witches’ broom disease in China

Chinese scholar tree (Sophora japonica) with witches’ broom symptoms was observed in Shandong Province in China. Phytoplasmas were detected in the diseased plants using 16S rDNA amplification with phytoplasma-specific universal primer pairs. On the basis of the results of 16S rDNA sequencing, virtual restriction fragment length polymorphism patterns and phylogenetic analyses, the phytoplasma found in S. japonica with witches’ broom symptoms was confirmed as a ‘Candidatus Phytoplasma ziziphi’-related strain belonging to the Elm yellows group 16SrV. This is the first report of ‘Ca. P. ziziphi’ infecting S. japonica plant with witches’ broom symptoms.     

Molecular analysis of ‘Candidatus Phytoplasma aurantifolia’ associated with phytoplasma diseases in Myanmar

During 2010 and 2011, typical phytoplasma disease symptoms such as little leaves, phyllody and witches’ brooms were observed on black gram, green gram, long bean, shaggy button weed and sesame plants from different regions of Myanmar. The symptomatic samples were analyzed by PCR using universal phytoplasma primers and characterized by sequencing 16S rRNA, ribosomal protein and translocase protein genes. Based on sequence and phylogenetic analysis of the three genes, the phytoplasmas associated with those plants belonged to members of ‘Candidatus Phytoplasma aurantifolia’. To our knowledge, black gram and shaggy button are new hosts for ‘Ca. P. aurantifolia’.     

‘Candidatus Phytoplasma australiense’ is the phytoplasma associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases

Sequence comparisons and phylogenetic analysis of the 16S rRNA genes and the 16S/23S spacer regions of the phytoplasmas associated with Australian grapevine yellows, papaya dieback and Phormium yellow leaf diseases revealed minimal nucleotide differences between them resulting in the formation of a monophyletic group. Therefore, along with Australian grapevine yellows, the phytoplasmas associated with Phormium yellow leaf and papaya dieback should also be considered as Candidatus Phytoplasma australiense.     

An abundant ‘Candidatus Phytoplasma solani’ tuf b strain is associated with grapevine,stinging nettle and Hyalesthes obsoletus

Bois noir (BN) associated with ‘Candidatus Phytoplasma solani’ (Stolbur) is regularly found in Austrian vine growing regions. Investigations between 2003 and 2008 indicated sporadic presence of the confirmed disease vector Hyalesthes obsoletus and frequent infections of bindweed and grapevine. Infections of nettles were rare. In contrast present investigations revealed a mass occurrence of H. obsoletus almost exclusively on stinging nettle. The high population densities of H. obsoletus on Urtica dioica were accompanied by frequent occurrence of ‘Ca. P. solani’ in nettles and planthoppers. Sequence analysis of the molecular markers secY, stamp, tuf and vmp1 of stolbur revealed a single genotype named CPsM4_At1 in stinging nettles and more than 64 and 90 % abundance in grapevine and H. obsoletus, respectively. Interestingly, this genotype showed tuf b type restriction pattern previously attributed to bindweed associated ‘Ca. P. solani’ strains, but a different sequence assigned as tuf b2 compared to reference tuf b strains. All other marker genes of CPsM4_At1 clustered with tuf a and nettle derived genotypes verifying distinct nettle phytoplasma genotypes. Transmission experiments with H. obsoletus and Anaceratagallia ribauti resulted in successful transmission of five different strains including the major genotype to Catharanthus roseus and in transmission of the major genotype to U. dioica. Altogether, five nettle and nine bindweed associated genotypes were described. Bindweed types were verified in 34 % of grapevine samples, in few positive Reptalus panzeri, rarely in bindweeds and occasionally in Catharanthus roseus infected by H. obsoletus or A. ribauti. ‘Candidatus Phytoplasma convolvuli‘(bindweed yellows) was ascertained in nettle and bindweed samples.     

Molecular diversity of ‘Candidatus Phytoplasma pyri’ isolates in Slovenia

A European quarantine organism ‘Candidatus Phytoplasma pyri’ causing devastating pear decline disease has been reported to affect pear trees in several European countries. In this study a multilocus sequence analysis was successfully used to gain detailed insight into the molecular diversity of thirty closely related ‘Candidatus Phytoplasma pyri’ isolates from different orchards in Slovenia. Among three genomic regions analyzed, the 16S/23S rRNA intergenic spacer region was the most conserved among Slovenian isolates with 99.7 % sequence identity, yielding only three distinct genotypes. On the other hand, five different genotypes were detected when analyzing secY and aceF genomic regions that shared sequence identity of 94.8 and 97.2 %, respectively. Six of the detected genotypes, specifically four in the secY region and one in each of the two other analyzed genomic regions, were unique for Slovenia. At least eight different haplotypes were found with multilocus sequence analysis, indicating high molecular diversity among Slovenian ‘Ca. P. pyri’ isolates. Haplotypes were clustered into two major clusters, separated by at least 45 mutations. No connection was established between haplotype occurrence and cultivar type.     

Desert rose witches’ broom disease associated with ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma aurantifolia’

In March 2011, witches’ brooms comprising many small shoots were observed on desert rose plants, Adenium obesum, in PyinOoLwin, Myanmar. The causal agent of the symptomatic leaves was diagnosed as a phytoplasma by polymerase chain reaction (PCR) analysis. Sequence analysis of the PCR product (1.8 kbp) showed the closest phylogenic relationships with members of the peanut witches’ broom phytoplasma group. Additionally, phylogenetic analyses revealed the phytoplasma is a member of ‘Candidatus Phytoplasma aurantifolia’. This is the first report of desert rose plant as a new host for ‘Ca. P. aurantifolia’.     

First report of ‘Candidatus Phytoplasma asteris’ infecting hydrangea showing phyllody in Japan

Phytoplasma-induced floral malformations such as virescence, phyllody, and proliferation were observed on hydrangeas in Gunma Prefecture, Japan. Phylogenetic analyses based on 16S rRNA, secY, groEL, and amp gene sequences indicated that the affected hydrangea plants were associated with phytoplasmas belonging to ‘Candidatus Phytoplasma asteris’, but not to ‘Ca. P. japonicum’, which occurs in hydrangeas showing phyllody in Japan. This is the first molecular evidence of an association of ‘Ca. P. asteris’ with hydrangea plants in Japan.     

Genetic diversity of Czech ‘Candidatus Phytoplasma mali’ strains based on multilocus gene analyses

In several European countries apple trees are affected by apple proliferation disease, which is usually associated with the presence of ‘Candidatus Phytoplasma mali’. During 2010, samples from several apple trees displaying proliferation symptoms were collected throughout the Czech Republic to verify identity of phytoplasmas detected in association with the disease. The majority of the 74 apple trees examined using molecular tools were positive for ‘Ca. P. mali’ presence. The 16S–23S ribosomal genes, the ribosomal protein genes and the nitroreductase and rhodonase like genes were then studied to verify phytoplasma strain variability on multigenic bases. Two RFLP profiles and correspondingly two genetic lineages were found in the PCR-amplified fragments covering the 16S–23S rDNA spacer region. ‘Ca. P. mali’ strains belonging to rpX-A subgroup were identified in the majority of the apple tree sampled, whereas phytoplasmas belonging to the rpX-B subgroup were distributed sporadically. The apple proliferation subtypes AP-15 and AT-2 exhibited nearly equal occurrence; the AT-1 subtype and a mixture of the two or all three of the AP subtypes were infrequently found. The PCR/RFLP results were confirmed by nucleotide sequence analyses of selected ‘Ca. P. mali’ strains.     

Important genetic diversity of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma solani’ related strains associated with bois noir grapevine yellows and planthoppers in Azerbaijan

Bois noir (BN) is an important grapevine yellows endemic to the Euro-Mediterranean basin caused by ‘Candidatus Phytoplasma solani’ (‘Ca. P. solani’), a non culturable plant pathogenic Mollicute. Bois noir symptoms could be associated with ‘Ca. P. solani’ in two Azerbaijanian vineyards where disease incidence and severity were recorded for five local Vitis vinifera cultivars. In order to gain insight into the epidemiology of Bois noir in Azerbaijan, ‘Ca. P. solani’ isolates infecting plants were characterized by multi-locus sequence analysis and their secY and stamp gene sequences compared to that of the strains detected in other plants and in local Cixiidae planthoppers. Genotypes were determined for two non-ribosomal house-keeping genes, namely tuf and secY, as well as two variable markers namely Stamp and mleP1 genes, that respectively encode the antigenic membrane protein AMP and a 2-Hydroxycarboxylate transporter. The Azerbaijanian BN phytoplasma isolates corresponded to three tufB and secY genotypes. A finer differentiation of Azerbaijanian ‘Ca. P. solani’ isolates was obtained with mleP1 as five different mleP1 genetic variants were found. Finally, Stamp gene allowed differentiating four new genotypes in grapevine among the 10 new Stamp genotypes detected in various plants in Azerbaijan. The preliminary survey for infected insects conducted in northern Azerbaijan, led to the identification of Hyalesthes obsoletus and Reptalus noahi as potential vectors for two ‘Ca. P. solani’ new genotypes phylogenetically distant from the known genetic clusters. Altogether these results indicate an important genetic diversity of BN phytoplasmas in Azerbaijan that certainly result from spread through local insect vectors.     

Detection and identification of a ‘<Emphasis Type="Italic">Candidatus</Emphasis> phytoplasma aurantifolia’-related strain (16SrII-A subgroup) associated with <Emphasis Type="Italic">corchorus aestuans</Emphasis> phyllody in China

In September 2015, a phyllody that is typical of phytoplasma infection was observed on Corchorus aestuans plants in Haikou, Hainan Province, China. Total DNA from symptomatic and asymptomatic plants was extracted for molecular diagnosis. On the basis of sequence analysis and phylogenetic trees based on 16S rDNA and rp genes, the phyllody phytoplasma was ascertained to be related to ‘Candidatus phytoplasma aurantifolia’. To the best of our knowledge, this is the first report of a phytoplasma infecting C. aestuans in the world.     

A novel phytoplasma associated with witches’ broom disease of Ligustrum ovalifolium in Turkey

California privet (Ligustrum ovalifolium Hassk.) plants exhibiting leaf yellowing, witches’ broom, dieback and decline symptoms were observed for two years (2010–2011) in three gardens at Adana region (Turkey). DNA isolated from symptomatic and healthy plants was used to amplify 16S rDNA fragments by direct and nested-PCR. Phytoplasmas were detected in 21 symptomatic plants, out of 30 samples collected, whilst no PCR amplifications were obtained from asymptomatic plants. BLAST analysis of the 16S rDNA showed that the phytoplasma found in L. ovalifolium from Turkey, denoted as Turkish Ligustrum witches’ broom phytoplasma (TuLiWB), most closely resembled members of group 16SrII (peanut witches’ broom group) and shared up to 92 % sequence identity. Based on in silico 16S rDNA RFLP analysis and automated calculation of the pattern similarity coefficient, TuLiWB showed molecular characteristics different from all previously described phytoplasma species to represent a new taxon. Similar indication also emerged from the phylogenetic tree which allocated it in a novel discrete subclade within the phytoplasma clade. This is the first report on the presence of a phytoplasma affecting L. ovalifolium and whether this novel phytoplasma is the same agent reported as a mycoplasma-like organism (MLO) and associated with witches’ broom disease of Ligustrum in Korea (1989) is yet to be determined.     

Molecular detection and identification of phytoplasma associated with pepper witches’ broom in China

Pepper witches’ broom (PWB) disease was observed in a field in Yangling, Shaanxi Province, China. The result of mechanical inoculation test for this disease was negative. Phytoplasma-like bodies were observed in ultrathin sections of petiole tissues of symptomatic samples. 16S rRNA gene and tuf gene of phytoplasma were amplified from the total DNA of symptomatic samples. Phylogeny analysis of the 16S rRNA gene and tuf gene suggested that the pepper witches’ broom associated phytoplasma belongs to the subgroup 16SrI-B, which was confirmed by the RFLP analysis of the 16S rRNA gene. The phytoplasma subgroup 16SrI-B was also detected in the vector Cicadella viridis trapped from the infected field. To our knowledge, this is the first report of 16SrI-B phytoplasma causing pepper witches’ broom in China.     

Seasonal colonisation of apple trees by ‘Candidatus Phytoplasma mali’ revealed by a new quantitative TaqMan real-time PCR approach

Apple proliferation (AP), caused by ‘Candidatus Phytoplasma mali’, is an economically important disease affecting many apple-growing areas in Europe. A new TaqMan real-time PCR assay was established for absolute quantification of ‘Ca. P. mali’ by using a single-copy gene of the host plant as a reference, which is amplified with the pathogen DNA in a single-tube reaction. Normalised estimates of phytoplasma concentration are ultimately expressed as the number of phytoplasma cells per host plant cell. The assay was used to monitor the ‘Ca. P. mali’ titre over the course of two growing seasons in roots and branches of symptomatic and asymptomatic but AP-positive apple trees. All 252 root samples from symptomatic and asymptomatic trees tested positive, with an average number of 59.8 ± 5.68 (standard error) and 55.1 ± 9.83 ‘Ca. P. mali’ per host cell, respectively. From the 378 shoot samples analysed, 81% of the symptomatic and only 20% of the asymptomatic samples were AP-positive with an average number of 9.4 ± 1.04 and 0.7 ± 0.13 ‘Ca. P. mali’ per host cell, respectively. This strengthens evidence that not the pathogen occurrence alone but the presence of a certain quantity of ‘Ca. P. mali’ in the aerial tree sections is involved in symptom expression. In addition, pronounced seasonality of the phytoplasma concentration was found, not only in branches, but also for the first time in roots of symptomatic and asymptomatic apple trees. Highest phytoplasma levels in roots were detected from December to May.     

Tuber transmission of ‘Candidatus Liberibacter solanacearum’ and its association with zebra chip on potato in New Zealand

Zebra chip, an emerging disease of potatoes, has recently been associated with ‘Candidatus Liberibacter solanacearum’ in New Zealand. The phloem-limited bacterium is known to be vectored by the tomato potato psyllid (Bactericera cockerelli). In this study, the role of tuber transmission in the spread of Ca. L. solanacearum was investigated by re-planting potato tubers infected with Ca. L. solanacearum in the absence of the psyllid. Nested PCR demonstrated that Ca. L. solanacearum could be transmitted from the mother tubers both to the foliage of growing plants and to progeny tubers, resulting in symptomatic and asymptomatic plants. Of 62 Ca. L. solanacearum-infected tubers four did not sprout symptomatic of zebra chip. A further two plants developed foliar symptoms associated with zebra chip during the growing season and died prematurely. Fifty-six of the infected tubers produced asymptomatic plants, although Ca. L. solanacearum was detected in the foliage of 39 of them indicative of transmission into asymptomatic progeny plants. At harvest, Ca. L. solanacearum was found in the daughter tubers of only five of the 39 asymptomatic plants, and only one of these plants was found to have zebra chip symptoms in the daughter tubers. Our results show that tuber transmission of Ca. L. solanacearum could play a role in the life cycle of this pathogen, providing a source for acquisition by Bactericera cockerelli and for movement of the pathogen to other regions of New Zealand via transport of seed tubers.     

Correction to: Haplotypes of ‘Candidatus Liberibacter solanacearum’ identified in Umbeliferous crops in Spain

European Journal of Plant Pathology -