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.     

Detection of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Phytoplasma brasiliense’ in a new geographic region and existence of two genetically distinct populations’

‘Candidatus Phytoplasma brasiliense’, a phytoplasma taxon associated with hibiscus witches’ broom disease was first described in 2001 in Brazil. In September 2007, a peach tree (Prunus persica) displaying yellowing symptoms reminiscent of phytoplasma infection was sampled in Guba region of Azerbaijan. A phytoplasma was detected in the diseased peach tree by nested PCR amplification of its 16S rDNA with universal primers for phytoplasmas. Phylogenetical analyses of the amplified 16S rDNA showed that the phytoplasma infecting the peach tree corresponded to ‘Ca. P. brasiliense’, a species never reported in Euro-Mediterranean area. To set up a detection assay, cloning of a ‘Ca. P. brasiliense’ DNA fragment was undertaken by comparative RAPD. The amplified dnaK-dnaJ genetic locus was used to design a nested PCR assay able to amplify all ‘Ca. P. brasiliense’ isolates of the subgroup 16SrXV-A without amplifying the related members of the group 16SrII. This assay also allowed confirming the first detection of ‘Ca. P. brasiliense’ in diseased basil collected in south Lebanon.     

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.     

Black band of Jew’s marrow caused by <Emphasis Type="Italic">Lasiodiplodia theobromae</Emphasis>

Stem rot and wilt of Jew’s marrow (nalta jute, Corchorus olitorius) were found on Is. Okinawa, Okinawa Prefecture, Japan, in March 2000. An anamorphic fungus, Lasiodiplodia theobromae was isolated repeatedly from the diseased plants and demonstrated to cause the disease. We coined the Japanese name “kurogare-byô” of Jew’s marrow for the present disease because it was new to Japan, although it had already been reported in India and Bangladesh as black band of the plant.     

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.     

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.     

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">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%.     

Report on the palm aphid, <Emphasis Type="Italic">Cerataphis brasiliensis</Emphasis> on ‘Kalparaksha’ coconut cultivar

Aggregations of palm aphid Cerataphis brasiliensis (Hempel) (Aphididae: Hemiptera) on the unfurled spindle leaf of ‘Kalparaksha’ variety of coconut, a selection from Malayan Green Dwarf, is reported for the first time. C. brasiliensis has not yet been observed from other coconut varieties, viz., West Coast Tall, Chowghat Orange Dwarf, Chowghat Green Dwarf, and Dwarf x Tall hybrids planted in the research farm of Central Plantation Crops Research Institute, Kayangulam, Kerala, India. Only the apterous form of the palm aphid was noticed in the semi-tall variety and a variety-induced emergence of C. brasiliensis is indicated under natural conditions. Due to the presence of two phoretic ant species, no natural predator was observed in the aphid colonies. Two sprays of dimethoate (0.05%) at an interval of 15–20 days were effective in suppression of the pest on Kalparaksha.     

Detection of <Emphasis Type="Italic">Ageratum enation virus</Emphasis> from cat’s whiskers (<Emphasis Type="Italic">Cleome gynandra</Emphasis> L.) with leaf curl symptoms in India

An association of a begomovirus with leaf curl symptoms on Cleome gynandra was detected using a polymerase chain reaction (PCR) with begomovirus-specific primers. Further, the complete DNA-A of the begomovirus was cloned and sequenced. BLAST analysis of the sequence data revealed 92–99% identities and close relationships with several isolates of Ageratum enation virus (AgEV); therefore, we identified the virus associated with leaf curl symptoms of C. gynandra as an isolate of AgEV. This report is the first on the detection of AgEV in plants of C. gynandra with leaf curl in India.     

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.     

Comparing p20’s RNA silencing suppressing activity among five phylogenetic groups of <Emphasis Type="Italic">Citrus Tristeza virus</Emphasis>

The p20 protein encoded by the Citrus Tristeza Virus (CTV) was previously identified as a RNA silencing suppressor. In this study, we analyzed the p20’s suppressing activity from five phylogenetic groups of CTV, using the co-infiltration assay of Green fluorescence protein (GFP) gene and the suppressor gene in 16C line Nicotiana benthamiana plants. Green fluorescence, GFP mRNA relative levels and GFP specific siRNAS were compared showing in most cases, only slight differences. Contrary to previous studies, the p20 suppressor was not able to impede neither short range nor systemic spreading of RNA silencing. The suppressor from the phylogenetic group 4 revealed a much reduced activity when compared with the others. At present we still don’t know whether this property is a characteristic of this group or an atypical feature due to a unique point mutation. The differences in the symptom type and intensity originated by isolates belonging to the phylogenetic groups assayed could not be related to differences to the p20 suppressor’s activity.     

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.     

Bois noir affects the yield and wine quality of <Emphasis Type="Italic">Vitis vinifera</Emphasis> L. cv. ‘Chardonnay’

The Bois noir (BN) disease induced by ‘Candidatus Phytoplasma solani’ (CPs) is common in European vineyards. Its damage has not been fully investigated, especially with regards to wine attributes. The impact of BN on yield, berry composition and wine characteristics of Vitis vinifera L. cv. ‘Chardonnay’ was therefore comprehensively characterized in a 3-year field experiment in Hungary, Eger winegrowing region. Additionally, the bindweed-related tuf-b1 genotype was identified to be involved in the BN pathosystem in the experimental vineyard. Infection of CPs tuf-b1 genotype resulted in severe yield loss, the average decrease in number of bunches and total yield per vine was 56.7% and 68.4%, respectively. Analyses of wines produced from grapes of BN infected vines revealed decreased alcohol, epicatechin and iron contents; and increased organic acids, titratable acidity, catechin and calcium contents. Sensory evaluation of these wines confirmed unfavourable characteristics, i.e. higher acidity, bitterness, and usually pinkish discolouration. Negative impact on berry composition and wine quality were pronounced in the vintage with favourable weather conditions for grapevine production, whereas the negative effects of BN infection were less prominent, even masked, in the vintages with unfavourable weather (wet and cool). To reduce BN-caused damage, the need for improved preventative and curative measures for BN disease is highlighted.     

Clove-transmissibility of <Emphasis Type="Italic">Pseudomonas salomonii</Emphasis>, the causal agent of ‘Café au lait’ disease of garlic

Bacterial blight of garlic, caused by Pseudomonas salomonii, results in leaf and sheath necrosis and sometimes leads to soft rot and plant death. The epidemiology of this bacterial disease, known as ‘Café au lait’ disease, is poorly understood and no resistant cultivars are currently available. To develop control strategies for this disease, we investigated principal sources of inoculum. The pathogen was isolated from bulbs from plants with typical vegetative symptoms of bacterial blight. Subsequent development of typical foliar symptoms on plantlets originating from symptomatic bulbs demonstrated transmission of the pathogen in the planting material. In one of three field experiments the contamination rate of planting stock influenced the disease incidence in field-grown garlic. The importance of planting stock as a source of inoculum was demonstrated here and should be evaluated relative to other potential sources such as crop debris, soil or alternate hosts in order to develop successful control strategies.     

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.     

Search for reservoirs of ‘<Emphasis Type="Italic">Candidatus</Emphasis> Liberibacter solanacearum’ and mollicutes in weeds associated with carrot and celery crops

Currently, the main arthropod vectored pathogens associated with carrot and celery crop diseases are ˋCandidatus Liberibacter solanacearum´, Spiroplasma citri and different phytoplasma species. Mitigation strategies require elucidating whether these pathogens survive in the weeds of these Apiaceae crops, which can act as reservoirs. Weed surveys were conducted in a vegetative cycle (April to October 2012) in the spontaneous vegetation that surrounded crops affected by ˋCa. L. solanacearum´, S. citri and/or phytoplasmas. Sixty-three species of 53 genera that belong to 23 botanical families were collected in the main carrot and celery Spanish production area. Species were identified, estimating coverage and abundance, and conserved in herbarium. Samples were analysed by nested-PCR with universal primers for phytoplasmas detection, and were sequenced for identification purposes; by conventional PCR for S. citri and real-time PCR for ˋCa. L. solanacearum´. The only detected pathogens were ˋCa. Phytoplasma trifolii´ (clover proliferation group 16Sr VI-A) in Amaranthus blitoides and Setaria adhaerens and ˋCa. P. solani´ (stolbur group 16Sr XII-A) in Convolvulus arvensis. These pathogens were also sporadically detected in celery or carrot crops. Unexpectedly, neither ˋCa. L. solanacearum´ nor S. citri was detected in the weed samples, despite the relatively high prevalence of these pathogens (less than 66 % and 25 %, respectively) in the surveyed plots. This suggests that weeds do not play an epidemiological role as reservoirs in the spread of such organisms in the studied region. The use of pathogen-free seed lots and the control of vectors are crucial for preventing the introduction and spread of these economical important pathogens to new areas.