The first detection of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma trifolii’ in <Emphasis Type="Italic">Rhododendron</Emphasis> hybridum

Four Rhododendron hybridum plants (from cvs Moravanka and Don Juan), all exhibited symptoms of shortened axillary shoots, reduced leaves with vein clearing and yellowing, undeveloped flowers, and general stunting in a rhododendron nursery garden in southern Bohemia in 2007. Electron microscopy examination of ultra-thin sections revealed the presence of numerous polymorphic phytoplasma-like bodies in the phloem tissue of leaf midribs and petioles. The phytoplasma etiology of this disease was further confirmed by polymerase chain reaction (PCR) using universal phytoplasma primers. Restriction fragment length polymorphism (RFLP) analysis of amplification products obtained with a R16F2/R16R2 primer pair from all symptomatic plants indicated the presence of phytoplasma from the 16SrVI-A subgroup. A detailed comparison of the amplified sequences and phylogenetic analysis confirmed that the phytoplasma belonged to the subgroup 16SrVI-A (clover proliferation phytoplasma group). This is the first report of the natural occurrence of ‘Candidatus Phytoplasma trifolii’ in plants of Rhododendron hybridum.     

First report of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma malaysianum’ associated with <Emphasis Type="Italic">Elaeocarpus</Emphasis> yellows of <Emphasis Type="Italic">Elaeocarpus zollingeri</Emphasis>

“Elaeocarpus yellows” (ELY) is a widely reported phytoplasma disease of Elaeocarpus zollingeri trees in Japan. The phytoplasma associated with ELY (ELY phytoplasma) had not been identified at the species level because its 16S rRNA sequence had yet to be reported. Here, we report the results of a sequence analysis based on 16S rRNA and secA gene sequences, which showed that the ELY phytoplasma is related to ‘Candidatus Phytoplasma malaysianum’. To our knowledge, this is the first report showing the occurrence of ‘Ca. P. malaysianum’ outside Malaysia and the infection of E. zollingeri by the phytoplasma.     

<Emphasis Type="Italic">Amaranthus</Emphasis> spp.: a new host of “<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma aurantifolia”

Mexico is considered to be one of the centers of origin of grain amaranth species. Recently, plants with abnormal anatomical features were observed in experimental fields established in Central Mexico. The most noticeable symptoms, which consisted of excessive stem and bud proliferation, mosaics and unusual coloration, suggested that they might be phytoplasma-induced disorders. Thus, different accessions of grain amaranth (Amaranthus hypochondriacus and A. cruentus) plants were subjected to polymerase chain reaction (PCR) analysis specifically designed to detect these pathogens. Two universal phytoplasma-specific primer pairs were tested in a nested PCR assay, with primer pair P1/tint (followed by primer pair R16F2/R16R2). Further DNA sequencing analysis of the resulting amplicons indicated that these phytoplasmas may be related to others already affecting important agricultural crops in Mexico, such as soybean. Data are presented that disclose the etiology of these syndromes by the use of molecular techniques. To the best of our knowledge, this finding constitutes the first report of a phytoplasma-related disease in grain amaranth.     

<Emphasis Type="Italic">Spiraea salicifolia:</Emphasis> a new plant host of “<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma ziziphi”-related phytoplasma

Spiraea salicifolia is widely grown in China as an ornamental plant, and its roots and young leaves have many medical uses. On the campus of Northwest A&F University, we observed diseased S. salicifolia plants that had yellowed, dwarfed, deformed leaves and other symptoms resembling diseases caused by phytoplasma. This study was aimed at determining the causal agent of the disease. On the basis of phytoplasma-specific DNA amplification by PCR, a phytoplasma infection of S. salicifolia was confirmed. The phytoplasma was related to “Ca. Phytoplasma ziziphi” according to RFLP and phylogenetic analyses. This report is the first of phytoplasma infection of S. salicifolia in China.     

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.     

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’.     

<Emphasis Type="Italic">In planta</Emphasis> multiplication and graft transmission of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter asiaticus’ revealed by Real-Time PCR

The bacterium Candidatus Liberibacter asiaticus (CLas) is associated with huanglongbing (HLB) in citrus in many countries. Despite the fact that many characteristics of the disease are known, the rate of multiplication of the bacterium within an infected tree is still poorly understood. To study this feature, we used the quantitative Real-Time polymerase chain reaction (Q-PCR) assay to follow and to quantify the multiplication of CLas in grafted infected young sweet orange plants. The rate of infection by grafting reached 100% at 120 days post-inoculation (dpi) showing that grafting could easily transmit CLas. A well-adjusted linear regression equation describing the bacterial growth in planta was obtained independently with measurements taken using repeated sampling in the same plant or different plants through the analysed period. The bacterial population, measured as copy number (CN) of the 16S rDNA target gene g⁻¹ of tissue, increased 10,000 times from 10³ at 30 dpi to approximately 10⁸ CN at 240 dpi indicating that CLas multiplication was fastest in young citrus plants. We observed a direct relationship between the concentration of pathogen and the expression of symptoms. Yellowed leaves or shoots, are commonly the first observed symptom of HLB, and were present in trees with a low amount of bacteria (10⁵ CN g⁻¹). Blotchy mottle symptoms were observed in trees with 10⁷ CN g⁻¹ of bacteria after 180 dpi. Buds taken from infected, but non-symptomatic branches were grafted on Rangpur lime and resulted in transmission rates ranging from 10 to 60%.     

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.     

Sex-specific probing behaviour of the carrot psyllid <Emphasis Type="Italic">Bactericera trigonica</Emphasis> and its implication in the transmission of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter solanacearum’

‘Candidatus Liberibacter solanacearum (Lso)’ is a pathogen of Solanaceae but also causes serious physiological disorders in carrots and celery (Apiaceae). In carrots, this pathogen is transmitted by the psyllids Bactericera trigonica and Trioza apicalis. How vector sex influences Lso transmission has not been yet elucidated. Here we report the probing behaviours of male and female B. trigonica and their impact on Lso titre transmitted, percentage of transmission, and symptoms produced on carrots when Lso is transmitted by males or females of B. trigonica. Vector sex affected the inoculation of Lso; our results suggest that females might inoculate higher Lso titres than males. However, the percentage of transmission was not affected by vector sex at a density of one or eight psyllids per plant. The number of yellow leaves and root weight were not different when Lso was transmitted by males or females at either of the psyllid densities tested, except for groups of females whose Lso transmission resulted in a higher number of yellow leaves than did Lso transmitted by groups of males. Electrical penetration graphs (EPG) showed that the proportion of individuals who reached phloem tissues was similar for males and females. However, EPGs also showed that females probed more times, ingested longer from phloem sieve elements and reached phloem tissues more frequently than did males during an 8-h inoculation access period (IAP). Our study shows that differences in probing behaviours between males and females of B. trigonica could modulate how Lso is inoculated by psyllids. These results highlight the importance of taking sex into consideration in psyllid studies of probing behaviour and bacterial transmission.     

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.     

Haplotypes of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter solanacearum’ identified in Umbeliferous crops in Spain

‘Candidatus Liberibacter solanacearum’ is a phloem-limited Gram-negative bacterium that causes serious damage to different crops of the botanical families Solanaceae and Apiaceae. Five haplotypes have been described: LsoA and LsoB are present in solanaceous crops in America and vectored by the tomato/potato psyllid Bactericera cockerelli; LsoC affects carrots from Northern and Central Europe, and is transmitted by the carrot psyllid Trioza apicalis; haplotypes LsoD and LsoE are present in Southern Europe and Morocco in carrot and celery, and are associated with the psyllid Bactericera trigonica. Thirty-four ‘Ca. L. solanacearum’ isolates were collected in six different regions of Spain from distinct Apiaceae hosts (carrot, celery, parsley and parsnip) in eight consecutive years and were analysed. Their haplotypes were determined by a sequence analysis of 16S ribosomal RNA, the 16S–26S ribosomal RNA intergenic spacer, and the 23S ribosomal RNA and rplJ and rplL genes. Both haplotypes LsoD and LsoE were found across Spain, and no host specificity appeared between these two haplotypes. This is the first report of ‘Ca. L. solanacearum’ associated with parsley and parsnip.     

Does <Emphasis Type="Italic">Puccinia hemerocallidis</Emphasis> regularly host-alternate between <Emphasis Type="Italic">Hemerocallis</Emphasis> and <Emphasis Type="Italic">Patrinia</Emphasis> plants in Japan?

Daylily rust fungus, Puccinia hemerocallidis, was proven to host-alternate between a wild daylily, Hemerocallis fulva var. longituba, and a patrinia, Patrinia villosa. No proof was obtained for the early belief that the fungus is pathogenic to plantainlilies, Hosta species, in addition to daylilies, Hemerocallis species. The fungus seems to alternate regularly between daylilies and patrinias in Japan because most daylily species are deciduous, and a vegetatively reproducing stage of the pathogen does not seem capable of successfully overwintering free of the living host tissue.     

Activity of <Emphasis Type="Italic">ß</Emphasis>-1,3-glucanase and <Emphasis Type="Italic">ß</Emphasis>-1,4-glucanase in two potato cultivars following challenge by the fungal pathogen <Emphasis Type="Italic">Alternaria solani</Emphasis>

Early blight of potato, caused by Alternaria solani, is a ubiquitous disease in many countries around the world. Our previous screening of several Iranian potato cultivars found that variation in resistance exists between two cultivars: ‘Diamond’ and ‘Granula’. Cultivar Diamond is more resistant to multiple isolates of A. solani when compared to cv. Granula. Furthermore, we have found that different pathogen isolates have varying degrees of infection. We monitored the activities of two pathogen-related (PR) glucanase proteins in Diamond and Granula in response to two isolates of A. solani with different degrees of virulence. ß-1,3-glucanase and ß-1,4-glucanase activities were recorded in healthy and diseased leaves of potatoes up to 10 days after inoculation. Their activities were found to be higher in diseased leaves when compared to those of uninfected leaves. Our data suggest that significantly reduced activities of theses enzymes in potato could be related to a lower degree of resistance or an increased ability of a more aggressive isolate to suppress PR protein expression.     

Recombination-like sequences in the upstream region of the phage-type DNA polymerase in ‘<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter asiaticus’

We determined and compared nucleotide sequences in the upstream region of the bacteriophage-type DNA polymerase in 15 Asian isolates of the citrus-greening bacterium ‘Candidatus Liberibacter asiaticus’ using the genomic information of American isolate Psy62. Some isolates had nucleotide changes and a large 156-bp deletion, which were concentrated in the putative bacteriophage genes ORF3 and ORF4 and corresponding intergenic region. Eight partial recombination-like patterns were observed in this variable region. Most were confirmed to have occurred between the original types 1 and 2, but a fragment, only several dozen nucleotides long and possibly derived from a third type, was also identified.     

Detection of <Emphasis Type="Italic">Phoma valerianellae</Emphasis> in lamb’s lettuce seeds

After the reappearance in Italy of a foliar disease of lamb’s lettuce (Valerianella olitoria L. Poll. syn. V. locusta L. Betcke) incited by Phoma valerianellae, we set out to measure the level of seed infection by this fungus, using a plating test, and to develop a molecular method for quick and reliable detection of the pathogen in seeds. All six samples of lamb’s lettuce seed tested were contaminated by P. valerianellae at levels of 0.6% to 15%. Surface disinfection of seeds did not eliminate the contamination and only reduced it to between 0.1% and 10%. The need for a sensitive, reliable and rapid diagnostic method for early identification of the fungus exists. We have developed a PCR-based method to identify the fungus in seeds. Variation within the internal transcribed spacer (ITS1, 5.8S sequences and ITS2) region of the rDNA (ITS) was used to characterize the P. valerianellae strains and to design specific primers within the ITS region.     

Haplotypes of “<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter solanacearum” suggest long-standing separation

Three haplotypes of the recently discovered bacterium species “Candidatus Liberibacter solanacearum” are described and related to geographic ranges. The first two are associated with Zebra Chip/Psyllid Yellows of potatoes and other solanaceous plants, vectored by the tomato/potato psyllid Bactericera cockerelli in North and Central America and New Zealand. The third is associated with diseased carrots in Finland and vectored by the carrot psyllid Trioza apicalis. The haplotypes are described by SNPs on the 16s rRNA, 16s/23s ISR and 50s rplJ and rplL ribosomal protein genes. These SNPs are inherited as a package across the three genes. Haplotype “a” has been found primarily from Honduras and Guatemala through western Mexico to Arizona and California, and in New Zealand. Haplotype “b” is currently known from eastern Mexico and northwards through Texas to south central Washington. These haplotypes show some range overlap in Texas, Kansas and Nebraska. The haplotypes are not yet known to elicit biological differences in the plant or insect hosts. These apparently stable haplotypes suggest separate bacterial populations of long standing.     

Effect of polyploidisation on the response of apple (<Emphasis Type="Italic">Malus</Emphasis> × <Emphasis Type="Italic">domestica</Emphasis> Borkh.) to <Emphasis Type="Italic">Venturia inaequalis</Emphasis> infection

Spinach is one of the most nutritious green-leaf vegetables. In the spinach production, diseases cause a significant loss in both yield and quality. Improving disease resistance is one of the major challenges in spinach breeding. Arabidopsis nucleoporin CONSTITUTIVE EXPRESSER OF PATHOGENESIS-RELATED GENES 5 (CPR5) functions as a negative regulator of plant cell death and immunity as cpr5 mutant exhibits spontaneous cell death and heightened immunity. In addition, CPR5 play a role in trichome development as the majority of cpr5 mutant trichomes are branchless whereas wild type trichomes are often three-branched. In the spinach genome, we identified a homolog of Arabidopsis CPR5, referred to as Spinacia oleracea CPR5 (SoCPR5). To investigate the function of SoCPR5, we introduced SoCPR5 into Arabidopsis cpr5 mutant. Our data showed that both spontaneous cell death and heightened immunity were suppressed in the SoCPR5-transgenic cpr5 mutants, verifying that SoCPR5 functions as its Arabidopsis counterpart in plant cell death and immunity. SoCPR5 also fully restored wild type trichome phenotype of the cpr5 mutant. Our study therefore indicates that the function of SoCPR5 is conserved between plant species and SoCPR5 can be applied for genetic manipulation of plant immunity in spinach.     

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.     

Detection and molecular characterisation of an alfalfa phytoplasma in Argentina that represents a new subgroup in the 16S rDNA Ash Yellows group(‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma fraxini’)

A phytoplasma infecting alfalfa crops was detected and characterised in Argentina, the Argentinean Alfalfa witches′-broom (ArAWB) phytoplasma. Typical witches′-broom symptoms were observed in diseased alfalfa plants from fields of the ‘Cuyo’ region in the Andean midwest. Pleomorphic bodies were observed by electron microscopy in sieve tubes of the diseased plants. The results obtained from the sequence homology, similarity coefficients derived from RFLP of the 16S rDNA and phylogenetic analysis led us to include this phytoplasma in the 16Sr VII (Ash Yellows) group. However, the ArAWB phytoplasma showed several differences when compared to other members of group 16Sr VII. The RFLP analysis of partial 16S rRNA gene of two ArAWB isolates, digested with 16 restriction enzymes, showed differences between the ArAWB and the reference strain (AshY1^T) in six enzyme patterns. Restriction patterns unique for the group and an exclusive HinfI restriction site were found in the ArAWB phytoplasma rDNA. Moreover, the similarity coefficients (0.92–0.86) were lower than those obtained among other group members. The significant differences detected suggested that this phytoplasma belonged to a subgroup different from those described so far. We propose therefore, that the ArAWB phytoplasma should be included in a new VII-C subgroup, closely related to the EriWB phytoplasma (VII-B) described in Brazil.