Samanta Paltrinieri

DNA barcoding for identification of 'Candidatus Phytoplasmas' using a fragment of the elongation factor Tu gene.

Background Phytoplasmas are bacterial phytopathogens responsible for significant losses in agricultural production worldwide. Several molecular markers are available for identification of groups or strains of phytoplasmas. However, they often cannot be used for identification of phytoplasmas from different groups simultaneously or are too long for routine diagnostics. DNA barcoding recently emerged as a convenient tool for species identification. Here, the development of a universal DNA barcode based on the elongation factor Tu (tuf) gene for phytoplasma identification is reported. Methodology/Principal Findings We designed a new set of primers and amplified a 420–444 bp fragment of tuf from all 91 phytoplasmas strains tested (16S rRNA groups -I through -VII, -IX through -XII, -XV, and -XX). Comparison of NJ trees constructed from the tuf barcode and a 1.2 kbp fragment of the 16S ribosomal gene revealed that the tuf tree is highly congruent with the 16S rRNA tree and had higher inter- and intra- group sequence divergence. Mean K2P inter−/intra- group divergences of the tuf barcode did not overlap and had approximately one order of magnitude difference for most groups, suggesting the presence of a DNA barcoding gap. The use of the tuf barcode allowed separation of main ribosomal groups and most of their subgroups. Phytoplasma tuf barcodes were deposited in the NCBI GenBank and Q-bank databases. Conclusions/Significance This study demonstrates that DNA barcoding principles can be applied for identification of phytoplasmas. Our findings suggest that the tuf barcode performs as well or better than a 1.2 kbp fragment of the 16S rRNA gene and thus provides an easy procedure for phytoplasma identification. The obtained sequences were used to create a publicly available reference database that can be used by plant health services and researchers for online phytoplasma identification.

Phytoplasmas Associated with Grapevine Yellows Disease in Chile

An extensive survey was performed from 2002 to 2006 to detect and identify phytoplasmas associated with Chilean grapevines. Nested polymerase chain reaction assays using phytoplasma universal primer pairs P1/P7 and R16F2n/R2 detected phytoplasmas in 34 out of the 94 samples tested (36%). Restriction fragment length polymorphism (RFLP) analyses, cloning, and sequencing allowed identification of phytoplasmas belonging to ribosomal subgroups 16SrI-B, 16SrI-C, 16SrVII-A, and 16SrXII-A. The 16SrVII-A phytoplasma represents a new finding in grapevine; moreover, variability of the RFLP profile was observed in some of the 16SrXII-A phytoplasmas, indicating possible new ribosomal subgroups. Mixed phytoplasma infections and infections of phytoplasmas together with one or more viruses also occurred.

Association of phytoplasmas and viruses with malformed clovers.

Plants ofTrifolium spp. exhibiting two different kinds of symptoms — phyllody associated with yellowing/reddening, and dwarf growth habit without floral abnormalities — were observed in several areas of the Czechia. Nested polymerase chain reaction (PCR) with phytoplasma specific primers, and restriction fragment length polymorphism (RFLP) analyses of 16SrDNA revealed that phyllody ofT. repens was associated with phytoplasmas belonging to the 16SrI-C subgroup. Similar symptoms inT. hybridum andT. pratense plants revealed the presence of phytoplasmas belonging to two subgroups: 16SrI-C and 16SrIII-B. Dwarf disease of cultivatedT. pratense plants was associated with more than one agent: 11 of 20 plants examined by PCR/RFLP analysis revealed the presence of phytoplasmas belonging to four distinct subgroups: 16SrI-B, 16SrI-C, 16SrIII-B and 16SrX-A. Moreover, two kinds of bacilliform virions were observed in ultrathin sections of 15T. pratense plants. Particles occurred mostly in the parenchymatous cells of vascular bundles and were located in the cytoplasm as aggregates within an extended network of membranous cisternae. Phytoplasmas and rhabdoviruses occurred singly, and both together or in co-presence with filamentous virus-like particles.

Correlation of bois noir disease with nettle and vector abundance in northern Italy vineyards

The phytoplasmas associated with bois noir (BN) tuf-type a are transmitted to grapevines by means of Hyalesthes obsoletus Signoret using Urtica dioica L. as the inoculum source. In the period 2003–2008, a research was carried out in six vineyards of northern Italy where a large amount of nettles in the ditches surrounding vineyards was detected. The aim was to establish the nettle control effects on the presence of H. obsoletus and new symptomatic grapevines. PCR and RFLP analyses showed the presence of the BN tuf-type a phytoplasmas in symptomatic grapevines of all vineyards and in the vector. Weed control strategies along ditches were always associated with decreases in nettle coverage and H. obsoletus numbers. A reduction in the percentage of new symptomatic grapevines occurred in all vineyards except for one vineyard where the least reduction in nettle and H. obsoletus was observed. The decrease in H. obsoletus population was significantly correlated with a decrease in nettles along ditches, and the reduction in H. obsoletus population was correlated with a decrease in incidence of new symptomatic grapevines compared with the incidence before the application of weed control measures. Nettle weeding can reduce the incidence of BN tuf-type a, but weed control costs and impacts need to be considered.

Leek Proliferation: A New Phytoplasma Disease in the Czech Republic and Italy

During the summer 1996, twelve of twenty-eight leek plants located in a garden near České Budějovice, South Bohemia exhibited symptoms typical of diseases associated with phytoplasmas. In summer 1998 similar symptoms were detected in leek plants in a field used for seed production located in Romagna, North Italy. In both cases the plants were established in the spring of the previous year. Plants showed flower abnormalities: stamen elongation, anther sterility, pistil proliferation, as well as poor, if any, seed production. Phytoplasma-like structures were detected by scanning and transmission electron microscopy in phloem sieve elements in the Czech diseased plants, but not in healthy ones. Nested-PCR amplifications of extracted DNA with phytoplasma-specific oligonucleotide primer pairs confirmed the presence of phytoplasmas in these plants at low concentrations. Restriction fragment length polymorphism analyses of amplified ribosomal sequences allowed the identification of detected phytoplasmas: all the samples from the Czech Republic contained aster yellows related phytoplasmas (16SrI-B) while in the Italian samples aster yellows related phytoplasmas (16SrI-B) together with stolbur related phytoplasmas (16SrXII-A) were identified. This is the first report of detection and identification of a phytoplasma disease of leek in the Czech Republic and Italy.

Nested PCR and RFLP analysis based on the 16S rRNA gene.

Current phytoplasma detection and identification methods are primarily based on nested polymerase chain reaction followed by restriction fragment length polymorphism analysis and gel electrophoresis. These methods can potentially detect and differentiate all phytoplasmas including those previously not described. The present protocol describes the application of this method for identification of phytoplasmas at 16S rRNA (16Sr) group and 16Sr subgroup levels.

Detection and identification of the coconut lethal yellowing phytoplasma in weeds growing in coconut farms in Côte d’Ivoire

Abstract Coconut farms located in the southern coast of Grand-Lahou in Côte d’Ivoire are severely affected by a lethal yellowing disease (CILY) associated with the group 16SrXXII-B, ‘Candidatus Phytoplasma palmicola’-related strains. Given the high prevalence of weed species on most of the farms, plants growing within and in the periphery of five selected coconut farms were assessed for the presence of the CILY phytoplasma to identify potential alternative hosts. A total of 396 plant samples belonging to 84 plant species and 35 botanical families were collected. Total DNA was extracted and tested by nested PCR with primers targeting the 16S rRNA and the translocation protein (secA) phytoplasma genes, and sequenced. Twenty samples from six plant species and five botanical families yielded PCR amplicons of the expected size, and both the secA and 16S rDNA sequences showed over 99% similarity with that of the Côte d’Ivoire lethal yellowing phytoplasma previously identified from coconut palms grown in Grand-Lahou coconut farms. Plant species from the families Poaceae (Paspalum vaginatum, Pennisetum pedicillatum), Verbenaceae (Stachytarpheta indica), Plantaginaceae (Scoparia dulcis), Phyllanthaceae (Phyllantus muellerianus) and Cyperacea (Diplacrum capitatum) were positive for the presence of the CILY phytoplasma, suggesting they may have epidemiological implications for disease spread in coconut farms in Grand-Lahou.

DNA Bar-Coding for Phytoplasma Identification

Phytoplasma identification has proved difficult due to their inability to be maintained in vitro. DNA barcoding is an identification method based on comparison of a short DNA sequence with known sequences from a database. A DNA barcoding tool has been developed for phytoplasma identification. While other sequence-based methods may be well adapted to identification of particular strains of phytoplasmas, often they cannot be used for the simultaneous identification of phytoplasmas from different groups. The phytoplasma DNA barcoding protocol in this chapter, based on the tuf and 16SrRNA genes, can be used to identify the following phytoplasma groups: 16SrI, 16SrII, 16SrIII, 16SrIV, 16SrV, 16SrVI, 16SrVII, 16SrIX, 16SrX, 16SrXI, 16SrXII, 16SrXV, 16SrXX, 16SrXXI.

Phytoplasma diseases on major crops in Vietnam

The nested-PCR technique was applied to detect and identify phytoplasmas associated with major crops in Vietnam including sugarcane, cassava and longan. Phylogenetic analysis confirmed that phytoplasmas associated with the sugarcane grassy shoot and white leaf diseases belong to the rice yellow dwarf group (16SrXI). In cassava showing witches’ broom symptoms a mixed infection of phytoplasmas related to ‘Candidatus Phytoplasma asteris’ (group 16SrI) and ‘Ca. P. aurantifolia’ (group 16SrII) was detected. In samples showing longan witches’ broom disease three phytoplasmas including ‘Ca. P. aurantifolia’ (group 16SrII), elm yellows (group 16SrV) and “stolbur” (group 16SrXII) were identified.

Occurrence and identification of grapevine phytoplasmas in main viticultural regions of Turkey

Intensive surveys were conducted in the main viticultural areas of Turkey in 2009-2010. Two hundred eighty nine symptomatic and 20 non-symptomatic plant samples were collected and subjected to nucleic acid extraction followed by PCR, nested PCR and RFLP analyses to detect phytoplasma presence and for their identification. The incidence rate of phytoplasma infection was 18.33% and the majority of the symptomatic grapevines were infected with grapevine yellows phytoplasmas belonging to 16SrXII-A subgroup (“bois noir”). Phytoplasmas of 16SrV group, aster yellows (16SrI-B) and pigeon pea witches’ broom (16SrIX) groups were also detected in the surveyed vineyards. Phytoplasma-associated infections were present more on wine grapevine cultivars (73.6%), such as Alphonse Lavallée, Alicante Bouschet, Chardonnay, Shiraz, Cabernet Sauvignon, Merlot, Sauvignon Blanc and Pinot Noir, compared to table grapes (26.4%), such as Bogazkere, Sirfani, Tahannebi and Emir.