A. Bertaccini

Phytoplasmas: diversity, taxonomy, and epidemiology

Phytoplasma associated diseases are spread worldwide, and in several cases are associated with severe epidemic of very often quarantine importance. These plant pathogens are prokaryotes belonging to the Mollicutes class since they lack a cell wall; up to now they were not cultivated in axenic culture therefore Koch postulates are only sometimes fulfilled by using alternative tools, such as graft or insect transmission. The possibility to design specific primers for highly conserved genes such as 16S ribosomal gene together with the use of molecular probes randomly cloned from phytoplasma genome, allowed discriminating and molecularly classifying them. Now a certain amount of knowledge is available that allow starting epidemiological studies in order to prevent further spreading of phytoplasma-associated diseases. In this paper molecular, biological and epidemiological characteristics of phytoplasma associated with important diseases worldwide are described.

Phytoplasma: ecology and genomic diversity.

ABSTRACT The recent development of molecular-based probes such as mono- and polyclonal antibodies, cloned phytoplasma DNA fragments, and phytoplasma-specific primers for polymerase chain reaction (PCR) has allowed for advances in detection and identification of uncultured phytoplasmas (formerly called mycoplasma-like organisms). Comprehensive phylogenetic studies based on analysis of 16S ribosomal RNA (rRNA) or both 16S rRNA and ribosomal protein gene operon sequences established the phylogenetic position of phytoplasmas as members of the class Mollicutes, and the revealed phylogenetic interrelationships among phytoplasmas formed a basis for their classification. Based on restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rRNA gene sequences, phytoplasmas are currently classified into 14 groups and 38 subgroups that are consistent with groups delineated based on phylogenetic analysis using parsimony of 16S rRNA gene sequences. In the past decades, numerous phyto-plasma strains associated with plants and insect vectors have been identified using molecular-based tools. Genomic diversity of phytoplasma groups appears to be correlated with their sharing common insect vectors, host plants, or both in nature. The level of exchange of genetic information among phytoplasma strains in a given group is determined by three-way, vector-phytoplasma-plant interactions. A putative mechanism for the creation of new ecological niches and the evolution of new ecospecies is proposed.

Improved detection methods for fruit tree phytoplasmas

Phytoplasmas infecting fruit trees are considered quarantine organisms in Europe and North America. Detection often is hampered by their extremely irregular distribution in host plants. A sensitive, specific and quick diagnostic test would be highly desirable for routine detection, mainly to avoid using infected planting material. PCR methods require tedious preparation of DNA; also, the available primers are highly specific and exhibit some homology to chloroplast and plastid DNA. To address these problems, we compared several DNA preparation protocols for purity of DNA, cost and time required. We also developed new primers using rDNA sequence information from an Austrian isolate of European Stone Fruit Yellows (ESFY). These primers operate at high annealing temperatures and, thus, increase the specificity and decrease the risk of false positives. The primers could reliably detect the European phytoplasmas (AP, ESFY and PD) within a collection of isolates maintained in micropropagated periwinkle. Thus, they are suitable as general primers for phytoplasma detection. The primers also can be used for strain identification by direct PCR followed by RFLP analysis as demonstrated with micropropagated fruit tree material. Finally, an IC-PCR method that uses the primers for AP detection was found very sensitive and suitable for large-scale testing of apple materialin vivo andin vitro.

Mixed infection of grapevines in northern Italy by phytoplasmas including 16S rRNA RFLP subgroup 16SrI-B strains previously unreported in this host.

Grapevine plants of cv. Chardonnay showing symptoms like those of flavescence doree disease in the field in Piemonte, Italy, contained phytoplasmas affiliated with two phylogenetically different 16S rRNA restriction fragment length polymorphism (RFLP) groups. These phytoplasmas were detected and identified by polymerase chain reaction (PCR) amplification of 16S rDNA and by RFLP analysis of amplified DNA as strains belonging to group 16SrI (aster yellows and related phytoplasmas) and group 16SrV (elm yellows and related phytoplasmas). Thirteen of 16 tested plants contained group l6SrI strains. Twelve contained strains belonging to subgroup 16SrI-G (Italian periwinkle virescence and related phytoplasmas), and one contained only a strain belonging to subgroup 16SrI-B (Maryland aster yellows and related phytoplasmas). One plant that contained a subgroup 16SrI-G phytoplasma strains also contained a strain belonging to group 16SrV. Three plants were doubly infected by subgroup 16SrI-G strains and strains belonging to subgroup 16SrI-B. These results indicate susceptibility of grapevines to infection by three distinct phytoplasmas, and reveal for the first time grapevine infection by subgroup 16SrI-B phytoplasmas and mixed infection of single grapevine plants by strains in two different subgroups in group 16SrI.

Identification of phytoplasmas in eggs, nymphs and adults of Scaphoideus titanus Ball reared on healthy plants

A survey for the presence of aster yellows‐related phytoplasmas in the different life stages of Scaphoideus titanus was carried out by means of PCR and nested‐PCR assays. Using a phytoplasma 16Srl group‐specific primer pair followed by RFLP analysis of amplified products, we were able to detect and identify phytoplasmas from eggs, newly hatched nymphs, fourth and fifth instar nymphs and adults reared on ten phytoplasma‐free Vicia faba seedlings. Two of these broad beans became infected, whereas PCR failed to detect phytoplasmas in the same ten plants before leafhopper rearing. These results suggest the possibility of transovarial transmission of aster yellows‐related phytoplasmas in S. titanus.

Phytoplasmas Associated with Elm Yellows: Molecular Variability and Differentiation from Related Organisms

Restriction fragment length polymorphism (RFLP) analyses were performed on polymerase chain reaction (PCR) amplimers of phytoplasmal DNA from eight samples obtained from Ulmus spp. (elms) affected by elm yellows (EY) in Italy and the United States, from Catharanthus roseus infected with strain EY1, and from five other plant species infected with phytoplasmas of the EY group sensu lato (group 16SrV). RFLP profiles obtained with restriction enzyme TaqI from ribosomal DNA amplified with primer pair P1/P7 differentiated elm-associated phytoplasmas from strains originally detected in Apocynum cannabinum, Prunus spp., Rubus fruticosus, Vitis vinifera, and Ziziphus jujuba. RFLP profiles obtained similarly with BfaI differentiated strains from A. cannabinum and V. vinifera from other phytoplasmas of group 16SrV. Elm-associated strains from within the United States had two RFLP patterns in ribosomal DNA based on presence or absence of an RsaI site in the 16S-23S spacer. Elm-associated phytoplasma strains from Italy were distinguished from those of American origin by RFLPs obtained with MseI in the same fragment of non-ribosomal DNA. Strain HD1, which was discovered in A. cannabinum associated with EY-diseased elms in New York State, was unique among the strains studied.

Characterization of a Phytoplasma Associated with Frogskin Disease in Cassava

Cassava frogskin disease (CFSD) is an economically important root disease of cassava (Manihot esculenta) in Colombia and other South American countries, including Brazil, Venezuela, Peru, Costa Rica, and Panama. The roots of severely affected plants are thin, making them unsuitable for consumption. In Colombia, phytoplasma infections were confirmed in 35 of 39 genotypes exhibiting mild or severe CFSD symptoms either by direct or nested polymerase chain reaction (PCR) assays employing ribosomal (r)RNA operon primer pairs. The CFSD-associated phytoplasmas were identified as group 16SrIII strains by restriction fragment length polymorphism (RFLP) and sequence analyses of amplified rDNA products, and results were corroborated by PCRs employing group 16SrIII-specific rRNA gene or ribosomal protein (rp) gene primers. Collectively, RFLP analyses indicated that CFSD strains differed from all phytoplasmas described previously in group 16SrIII and, on this basis, the strains were tentatively assigned to new ribosomal and ribosomal protein subgroups 16SrIII-L and rpIII-H, respectively. This is the first molecular identification of a phytoplasma associated with CFSD in cassava in Colombia.

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.

Identification and molecular characterization of the phytoplasma associated with peach rosette-like disease at the Canadian Clonal Genebank based on the 16S rRNA gene analysis

Abstract Peach trees exhibiting peach rosette-like disease symptoms and infected by a phytoplasma of group 16SrI ‘Candidatus Phytoplasma asteris’ at the Canadian Clonal Genebank were further tested for the pathogen characterization based on the 16S rRNA gene. Nested PCR with phytoplasma universal primers R16mF2/R1 and R16F2n/R2 resulted in amplification of products of approximately 1.25 kb from all four symptomatic trees tested. Virtual RFLP of the R16F2n/R2 sequenced amplicons with selected restriction endonucleases showed unique RFLP patterns when compared to the described 16SrI phytoplasma subgroups; these data were confirmed by phylogenetic analyses. The phytoplasma was therefore assigned as a member of a new 16SrI subgroup (16SrI-W). Results represent the first report of a new phytoplasma 16SrI subgroup infecting peach in Canada, and provide a valuable tool for further epidemiological studies on this phytoplasma in peach.

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.