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.     

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.     

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.     

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 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.     

‘Candidatus Phytoplasma aurantifolia’-related strain associated with tomato yellows disease in China

A new disease of tomato plants with typical phytoplasma disease symptoms such as stunting, yellows, auxiliary shoot proliferation and phyllody was observed in Yunnan Province, southwest China in 2011. By a nested-PCR, phytoplasma were detected using the phytoplasma universal primers specific for 16S rDNA. The results of the 16S rDNA sequencing, computer-simulated RFLP patterns and phylogenetic analysis indicated that the phytoplasma associated with the diseased tomato plants belongs to subgroup A of the peanut witches’-broom group. This is the first report of a 16SrII-A phytoplasma associated with a new tomato disease in China. This new disease was named tomato yellows.     

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.     

‘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.     

Molecular diversity of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma mali’ and ‘<Emphasis Type="Italic">Ca</Emphasis>. P. prunorum’ in orchards in Slovenia

The plant parasitic nematode Longidorus poessneckensis found in Austria, the Czech Republic, Germany, Poland and the Slovak Republic was molecularly characterized. Mitochondrial genes encoding cytochrome c oxidase subunit I (COI) and nicotinamide dehydrogenase subunit 4 (nad4), the D2 and D3 expansion segments of 28S rRNA and Internal transcribed spacer 1 (ITS1) rRNA were sequenced for 16 L. poessneckensis populations. Six haplotypes of COI and five haplotypes of nad4 were determined. Nucleotide intraspecific variation was up to 17.1% for the partial sequenced COI gene and up to 17.7% for the partial sequenced nad4 gene, the latter being the highest up to date known intraspecific variation in this genus. The analyses of multiple amino acid sequence alignments of mitochondrial genes revealed low variability (0–2.4%) for COI gene and high divergence (0–7.6%) for nad4 gene. Intraspecific sequence diversity for the D2-D3 of 28S rRNA gene was up to 1.2% and for ITS1 rRNA gene was up to 1.6%. It has been hypothesized, that during the Last Glacial Maximum, L. poessneckensis populations probably persisted in refuge areas in the Carpathian Mountains and subsequently expanding from these areas and colonizing other European regions.     

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.     

Evaluation of efficiency of hemi-nested PCR assay for the detection of ‘Candidatus Liberibacter’ infecting citrus

Journal of Plant Diseases and Protection - The present study reports the development and evaluation of a hemi-nested polymerase chain reaction (hnPCR) assay for the efficient detection of...     

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.     

Note: Molecular identification of ‘Candidatus phytoplasma asteris’ inducing histological anomalies inSilene nicaeensis

Numerous plants ofSilene nicaeensis having symptoms resembling those associated with the presence of phytoplasmas were observed in an extensive coastal area in the south of Italy. Microscopic observation showed histological abnormalities in the organization of tissues in symptomatic plants, and molecular tests, including PCR/RFLP analyses and nucleic acid sequencing, revealed the presence of phytoplasmas belonging to the aster yellows group (‘Candidatus phytoplasma asteris’). This is the first report of phytoplasma infection inS. nicaeensis, a wild species that colonizes the Calabrian coast. http://www.phytoparasitica.org posting June 12, 2008     

Genetic diversity of ‘<Emphasis Type="Italic">Cadidatus</Emphasis> Liberibacter solanacearum’ strains in the United States and Mexico revealed by simple sequence repeat markers

‘Candidatus Liberibacter solanacearum’ is associated with the Zebra Chip (ZC) disorder of potatoes. A panel of eight simple sequence repeat (SSR) markers was developed and used to genetically characterize ‘Ca. L. solanacearum’ strains obtained from ZC-affected potato plants in the United States and Mexico. The multilocus SSR markers in this study effectively differentiated genotypes and estimated genetic diversity of ‘Ca. L. solanacearum’ strains. Genotype assignment analyses identified two major lineages of ‘Ca. L. solanacearum’ in the North American populations while only one lineage type was identified in Mexican population. No clear genetic structure was found among haplotypes based on geographical proximity or host. The high resolution power of the SSR marker system developed in this study provides a useful tool for genotyping closely related strains and tracking sources of the pathogen. Genotype information combined with epidemiological data will advance knowledge of ZC disease and will facilitate development of effective disease management.     

Detection of ‘Candidatus Liberibacter asiaticus’ in citrus by concurrent tissue print-based qPCR and immunoassay

‘Candidatus Liberibacter asiaticus’ (CLas) is associated with the most destructive disease of citrus, huanglongbing (HLB). The most widely used methods for detection of CLas are PCR-based and require purification of DNA from plant samples. Elution of DNA from tissue prints made on nitrocellulose membranes followed by qPCR (TPE-qPCR) was compared to DNA extraction of plant tissue followed by PCR (X-PCR) by testing the same tissue samples. The former estimated a higher CLas population in tissue prints than the latter (t-test; P = 0.009). All extracts prepared for TPE-qPCR throughout the experiment were also tested by conventional PCR and 80.8% were identified as positive. A similar set of stem and petiole tissue samples was tested by TPE-qPCR and immunoassay. Although the detection rate by TPE-qPCR was higher than by immunoassay, about 6% of tissue prints were positive by immunoassay but not by TPE-qPCR. Thus, a higher detection rate would be achieved by combining TPE-qPCR with immunoassay. Significant differences were observed in the performance of nitrocellulose membranes from different manufacturers in these assays. Immunotissue prints showed that the spatial distribution pattern of CLas infection varied widely from one sample to another, but the patterns were highly correlated among serial sections from the same sample, suggesting that CLas preferentially colonizes adjacent phloem cells in a vertical rather than horizontal direction.     

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

European Journal of Plant Pathology -     

Molecular typing of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma mali’ and epidemic history tracing by a combined T-RFLP/VNTR analysis approach

Apple proliferation caused by ‘Candidatus Phytoplasma mali’ is a disease of apple trees gaining increasing importance in Europe. The present study describes a high-throughput method for simultaneous typing of ‘Ca. P. mali’ at two genetic loci. This novel approach combines terminal restriction fragment length polymorphism (T-RFLP) analysis of a putative rhodanese-like protein gene and the analysis of the variable number of tandem repeats (VNTR) of the ribosomal protein L22 gene. The typing approach was applied to analyse a collection of DNA isolates from 310 apple trees tested positive for ‘Ca. P. mali’. Samples were taken between 2002 and 2010 in South Tyrol (Northern Italy). In addition, 15 samples of Cacopsylla melanoneura and 19 of C. picta were typed. Seven combined genetic profiles were found in the samples of infected apple trees: AT-2/rpX-A (81.0%), AT-1/rpX-D (8.4%), AT-1/rpX-E (4.2%), AT-1/rpX-A (3.2%), AT-1/rpX-B (1.6%), AT-1/rpX-C (1.0%) and AP/rpX-A (0.3%), and one mixed infection AP + AT-1/rpX-A + rpX-D (0.3%). Subtype rpX-E was discovered for the first time. In C. melanoneura samples the most frequent subtype was AT-1/rpX-E, followed by AT-1/rpX-D and AT-1/rpX-C. All C. picta samples displayed subtype AT-2/rpX-A. Analysis of the temporal distribution of subtype frequencies in apple trees revealed that exclusively subtype AT-1 in combination with four rpX subtypes was present in South Tyrol in the period from 2002 to 2004. From 2006 onwards subtype AT-2/rpX-A became dominant with an average frequency of 90%. The data obtained suggest that there may be a co-adaptation of particular ‘Ca. P. mali’ subtypes with different insect vector species.     

Multilocus sequence analysis supports a low genetic diversity among ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma australasia’ related strains infecting vegetable crops and periwinkle in Egypt

‘Candidatus Phytoplasma australasia’ causes important damages to the Egyptian vegetable crop production. A prerequisite for controlling the different diseases it causes to eggplant, tomato and squash, is to trace its propagation pathways. To allow the differentiation of ‘Ca. P. australasia’ strains, a multilocus sequence analysis protocol was developed. Four conserved phytoplama genes namely tuf, secY, dnaK and dppA, were selected among the CDS of a ‘Ca. P. aurantifolia’ genome draft. The corresponding genes were PCR amplified from tomato, eggplant and squash collected in 2010 from the governorates Sharkia, Elmynia and Beni sueif, as well as from Catharanthus roseus periwinkles collected in 2013 from Kafrelsheikh governorate. Sequence comparisons showed no diversity among the Egyptian isolates of ‘Ca. P. australasia’ that also constitute a distinct cluster within the 16SrII-D taxonomic subgroup. This low diversity supports a common epidemiology for the different diseases affecting vegetable crops and periwinkle in Egypt and suggests that future investigations on insect vector should focus on polyphagous leafhoppers.