Detection and identification of Bogia coconut syndrome phytoplasma from seed-associated tissues and seedlings of coconut (Cocos nucifera) and betel nut (Areca catechu).

Evidence for seed transmission of phytoplasmas has grown in several pathosystems including coconut (Cocos nucifera). Bogia coconut syndrome (BCS) is a disease associated with the lethal yellowing syndrome associated with the presence of 'Candidatus Phytoplasma noviguineense' that affects coconut, betel nut (Areca catechu) and bananas (Musa spp.) in Papua New Guinea. Coconut and betel nut drupes were sampled from BCS-infected areas in Papua New Guinea, dissected, the extracted nucleic acid was used in polymerase chain reaction (PCR), and loop mediated isothermal amplification (LAMP) used to check for presence of phytoplasma DNA. In a second study, drupes of both plant species were collected from multiple field sites and grown in insect-proof cages. Leaf samples taken at 6 months were also tested with PCR and LAMP. The studies of dissected coconut drupes detected phytoplasma DNA in several tissues including the embryo. Drupes from betel nut tested negative. Among the seedlings, evidence of possible seed transmission was found in both plant species. The results demonstrate the presence of 'Ca. P. noviguineense' in coconut drupes and seedlings, and in seedlings of betel nut; factors that need to be considered in ongoing management and containment efforts.

Use of the 23S rRNA gene as a target template in the universal loop-mediated isothermal amplification (LAMP) of genomic DNA from phytoplasmas.

Plant-pathogenic bacteria cause numerous diseases in host plants and can result in serious damage. Timely and accurate diagnostic techniques are, therefore, crucial. While advances in molecular techniques have led to diagnostic systems able to distinguish known plant pathogens at the species or strain level, systems covering larger categories are mostly lacking. In this study, a specific and universal LAMP-based diagnostic system was developed for phytoplasmas, a large group of insect-borne plant-pathogenic bacteria that cause significant agricultural losses worldwide. Targeting the 23S rRNA gene of phytoplasma, the newly designed primer set CaPU23S-4 detected 31 'Candidatus Phytoplasma' tested within 30 min. This primer set also showed high specificity, without false-positive results for other bacteria (including close relatives of phytoplasmas) or healthy plants. The detection sensitivity was ~10,000 times higher than that of PCR methods for phytoplasma detection. A simple, rapid method of DNA extraction, by boiling phytoplasma-infected tissues, was developed as well. When used together with the universal LAMP assay, it enabled the prompt and accurate detection of phytoplasmas from plants and insects. The results demonstrate the potential of the 23S rRNA gene as a versatile target for the LAMP-based universal detection of bacteria at the genus level and provide a novel avenue for exploring this gene as molecular marker for phytoplasma presence detection.IMPORTANCEPhytoplasmas are associated with economically important diseases in crops worldwide, including lethal yellowing of coconut palm, "flavescence dorée" and "bois noir" of grapevine, X-disease in stone fruits, and white leaf and grassy shoot in sugarcane. Numerous LAMP-based diagnostic assays, mostly targeting the 16S rRNA gene, have been reported for phytoplasmas. However, these assays can only detect a limited number of 'Candidatus Phytoplasma' species, whereas the genus includes at least 50 of these species. In this study, a universal, specific, and rapid diagnostic system was developed that can detect all provisionally classified phytoplasmas within 1 h by combining the LAMP technique targeting the 23S rRNA gene with a simple method for DNA extraction. This diagnostic system will facilitate the on-site detection of phytoplasmas and may aid in the discovery of new phytoplasma-associated diseases and putative insect vectors, irrespective of the availability of infrastructure and experimental resources.

Phytoplasma diseases of plants: molecular diagnostics and way forward.

Phytoplasmas are obligate phytopathogenic bacteria associated with devastating diseases in hundreds of crops across the world. They have been responsible for huge economic losses in many crop plants for decades now. Isolation and establishment of axenic culture of phytoplasma in complex media is a recent progress in phytoplasma research. Earlier methods for phytoplasma disease detection included symptom profiling, microscopy, serology and dodder transmission studies. With advancement in the field of molecular biology, phytoplasma diagnostics and characterisation witnessed radical improvement. Starting from PCR amplification which often necessities a nested PCR on account of low titre of phytoplasmas, to the closed tube quantitative PCR assays and then the ddPCR, an array of diagnostics have been developed for phytoplasma. The isothermal diagnostic platforms are the latest addition to this and the Loop Mediated Isothermal Amplification (LAMP) assay has been applied for the detection of phytoplasma from several hosts. The futuristic approach in phytoplasma detection will be very likely provided by an integration of nanotechnology and molecular diagnostics. Phytoplasma disease management majorly relies on early detection, vector control, use of disease free planting materials and cultivation of resistant varieties. Hence understanding the molecular mechanism of phytoplasma-host interaction is as important as timely and accurate detection, in the management of phytoplasma diseases. Further, the changing climatic scenario and global warming may lead to an upsurge in the phytoplasma diseases spread and severity across the world, making disease management even more challenging.

'Candidatus Phytoplasma dypsidis', a novel taxon associated with a lethal wilt disease of palms in Australia.

A phytoplasma was initially detected in Dypsis poivriana by nested and real-time PCR from the botanical gardens in Cairns, Queensland, Australia in 2017. Further surveys in the Cairns region identified phytoplasma infections in eight additional dying ornamental palm species (Euterpe precatoria, Cocos nucifera, Verschaffeltia splendida, Brassiophoenix drymophloeodes, Burretiokentia hapala, Cyrtostachys renda, Reinhardtia gracilis, Carpoxylon macrospermum), a Phoenix species, a Euterpe species and two native palms (Archontophoenix alexandrae). Analysis of 16S rRNA gene sequences showed that this phytoplasma is distinct as it shared less than 97.5 % similarity with all other 'Candidatus Phytoplasma' species. At 96.3 % similarity, the most closely related formally described member of the provisional 'Ca. Phytoplasma' genus was 'Ca. Phytoplasma noviguineense', a novel taxon from the island of New Guinea found in monocotyledonous plants. It was slightly more closely related (96.6-96.8 %) to four palm-infecting strains from the Americas, which belong to strain group 16SrIV and which have not been assigned to a formal 'Candidatus Phytoplasma' species taxon. Phylogenetic analysis of the 16S rRNA gene and ribosomal protein genes of the phytoplasma isolate from a dying coconut palm revealed that the phytoplasma represented a distinct lineage within the phytoplasma clade. As the nucleotide identity with other phytoplasmas is less than 97.5 % and the phylogenetic analyses show that it is distinct, a novel taxon 'Candidatus Phytoplasma dypsidis' is proposed for the phytoplasma found in Australia. Strain RID7692 (GenBank accession no. MT536195) is the reference strain. The impact and preliminary aspects of the epidemiology of the disease outbreak associated with this novel taxon are described.

The agent associated with blue dwarf disease in wheat represents a new phytoplasma taxon, 'Candidatus Phytoplasma tritici'.

Wheat blue dwarf (WBD) is one of the most economically damaging cereal crop diseases in northwestern PR China. The agent associated with the WBD disease is a phytoplasma affiliated with the aster yellows (AY) group, subgroup C (16SrI-C). Since phytoplasma strains within the AY group are ecologically and genetically diverse, it has been conceived that the AY phytoplasma group may consist of more than one species. This communication presents evidence to demonstrate that, while each of the two 16 rRNA genes of the WBD phytoplasma shares >97.5 % sequence similarity with that of the 'Candidatus Phytoplasma asteris' reference strain, the WBD phytoplasma clearly represents an ecologically separated lineage: the WBD phytoplasma not only has its unique transmitting vector (Psammotettix striatus) but also elicits a distinctive symptom in its predominant plant host (wheat). In addition, the WBD phytoplasma possesses molecular characteristics that further manifest its significant divergence from 'Ca. P. asteris'. Such molecular characteristics include lineage-specific antigenic membrane proteins and a lower than 95 % genome-wide average nucleotide identity score with 'Ca. P. asteris'. These ecological, molecular and genomic evidences justify the recognition of the WBD phytoplasma as a novel taxon, 'Candidatus Phytoplasma tritici'.

'Candidatus Phytoplasma stylosanthis', a novel taxon with a diverse host range in Australia, characterised using multilocus sequence analysis of 16S rRNA, secA, tuf, and rp genes.

In Australia, Stylosanthes little leaf (StLL) phytoplasma has been detected in Stylosanthes scabra Vogel, Arachis pintoi Krapov, Saccharum officinarum L., Carica papaya L., Medicago sativa L., and Solanum tuberosum L. The 16S rRNA gene sequence of StLL phytoplasma strains from S. scabra, C. papaya, S. officinarum and S. tuberosum were compared and share 99.93-100 % nucleotide sequence identity. Phylogenetic comparisons between the 16S rRNA genes of StLL phytoplasma and other 'Candidatus Phytoplasma' species indicate that StLL represents a distinct phytoplasma lineage. It shares its most recent known ancestry with 'Ca. Phytoplasma luffae' (16SrVIII-A), with which it has 97.17-97.25 % nucleotide identity. In silico RFLP analysis of the 16S rRNA amplicon using iPhyClassifier indicate that StLL phytoplasmas have a unique pattern (similarity coefficient below 0.85) that is most similar to that of 'Ca. Phytoplasma luffae'. The unique in silico RFLP patterns were confirmed in vitro. Nucleotide sequences of genes that are more variable than the 16S rRNA gene, namely tuf (tu-elongation factor), secA (partial translocation gene), and the partial ribosomal protein (rp) gene operon (rps19-rpl22-rps3), produced phylogenetic trees with similar branching patterns to the 16S rRNA gene tree. Sequence comparisons between the StLL 16S rRNA spacer region confirmed previous reports of rrn interoperon sequence heterogeneity for StLL, where the spacer region of rrnB encodes a complete tRNA-Isoleucine gene and the rrnA spacer region does not. Together these results suggest that the Australian phytoplasma, StLL, is unique according to the International Organization for Mycoplasmology (IRPCM) recommendations. The novel taxon 'Ca. Phytoplasma stylosanthis' is proposed, with the most recent strain from a potato crop in Victoria, Australia, serving as the reference strain (deposited in the Victorian Plant Pathology Herbarium as VPRI 43683).

'Candidatus Phytoplasma sacchari', a novel taxon - associated with Sugarcane Grassy Shoot (SCGS) disease.

Sugarcane Grassy Shoot (SCGS) disease is known to be related to Rice Yellow Dwarf (RYD) phytoplasmas (16SrXI-B group) which are found predominantly in sugarcane growing areas of the Indian subcontinent and South-East Asia. The 16S rRNA gene sequences of SCGS phytoplasma strains belonging to the 16SrXI-B group share 98.07 % similarity with 'Ca. Phytoplasma cynodontis' strain BGWL-C1 followed by 97.65 % similarity with 'Ca. P. oryzae' strain RYD-J. Being placed distinctly away from both the phylogenetically related species, the taxonomic identity of SCGS phytoplasma is unclear and confusing. We attempted to resolve the phylogenetic positions of SCGS phytoplasma based on the phylogenetic analysis of 16S rRNA gene (>1500 bp), nine housekeeping genes (>3500 aa), core genome phylogeny (>10 000 aa) and OGRI values. The draft genome sequences of SCGS phytoplasma (strain SCGS) and Bermuda Grass White leaf (BGWL) phytoplasma (strain LW01), closely related to 'Ca. P. cynodontis', were obtained. The SCGS genome was comprised of 29 scaffolds corresponding to 505 173 bp while LW01 assembly contained 21 scaffolds corresponding to 483 935 bp with the fold coverages over 330× and completeness over 90 % for both the genomes. The G+C content of SCGS was 19.86 % while that of LW01 was 20.46 %. The orthoANI values for the strain SCGS against strains LW01 was 79.42 %, and dDDH values were 22. Overall analysis reveals that SCGS phytoplasma forms a distant clade in RYD group of phytoplasmas. Based on phylogenetic analyses and OGRI values obtained from the genome sequences, a novel taxon 'Candidatus Phytoplasma sacchari' is proposed.

Genetic variation, phylogenetic relationship and spatial distribution of 'Candidatus Phytoplasma ulmi' strains in Germany.

A recent survey in Germany revealed the wide presence of 'Candidatus Phytoplasma ulmi' in native elm stands. Accessions were studied for their genetic variability and phylogenetic relationship based on the conserved groEL and the variable imp gene. While the groEL sequences revealed a high intraspecific homology of more than 99%, the homology of the imp gene dropped to 71% between distantly related sequences. Twenty-nine groEL and 74 imp genotypes were distinguished based on polymorphic sites. Phylogenetic analysis of the groEL gene clustered all 'Ca. P. ulmi' strains and separated them from related phytoplasmas of the 16SrV group. The inferred phylogeny of the imp gene resulted in a different tree topology and separated the 'Ca. P. ulmi' genotypes into two clusters, one closely related to the flavescence dorée phytoplasma strain FD-D (16SrV-D), the other affiliated with the flavescence dorée phytoplasma strains FD-C and FD70 and the alder yellows phytoplasma (16SrV-C). In both phylograms, 'Ca. P. ulmi' genotypes from Scots elm trees formed a coherent cluster, while genotypes from European white elms and field elms grouped less strictly. The regional distribution pattern was congruent for some of the groEL and imp genotypes, but a strict linkage for all genotypes was not apparent.

Detection of Lethal Bronzing Disease in Cabbage Palms (Sabal palmetto) Using a Low-Cost Electronic Nose.

Lethal Bronzing Disease (LB) is a disease of palms caused by the 16SrIV-D phytoplasma. A low-cost electronic nose (eNose) prototype was trialed for its detection. It includes an array of eight Taguchi-type (MQ) sensors (MQ135, MQ2, MQ3, MQ4, MQ5, MQ9, MQ7, and MQ8) controlled by an Arduino NANO® microcontroller, using heater voltages that vary sinusoidally over a 2.5 min cycle. Samples of uninfected, early symptomatic, moderate symptomatic, and late symptomatic infected palm leaves of the cabbage palm were processed and analyzed. MQ sensor responses were subjected to a 256 element discrete Fourier transform (DFT), and harmonic component amplitudes were reviewed by principal component analysis (PCA). The experiment was repeated three times, each showing clear evidence of differences in sensor responses between the samples of uninfected leaves and those in the early stages of infection. Within each experiment, four groups of responses were identified, demonstrating the ability of the unit to repeatedly distinguish healthy leaves from diseased ones; however, detection of the severity of infection has not been demonstrated. By selecting appropriate coefficients (here demonstrated with plots of MQ5 Cos1 vs. MQ8 Sin3), it should be possible to build a ruleset classifier to identify healthy and unhealthy samples.

Development of three duplex real-time RT-PCR assays for the sensitive and rapid detection of a phytoplasma and five viral pathogens affecting stone fruit trees.

Three duplex real-time reverse-transcription polymerase chain reaction (real-time RT-PCR) assays based on TaqMan chemistry, were developed for the simultaneous detection and specific quantification of apple chlorotic leafspot virus (ACLSV), plum pox virus (PPV), prunus necrotic ringspot virus (PNRSV), prune dwarf virus (PDV), peach latent mosaic viroid (PLMVd) and the European stone fruit yellows (ESFY) phytoplasma, which are considered among the most important pathogens affecting stone fruit trees. The quantitative RT-PCR (RT-qPCR) assays were optimized using RNA transcripts (linearized plasmid was used for the assay optimization of the ESFY phytoplasma) of known concentrations. No differences in sensitivity were recorded between the duplex and singleplex RT-qPCR assays. The amplification efficiency of the duplex assays reached 91.1-95.8%, while the linear range of quantification was from 20 to 2 × 107 RNA/linearized plasmid transcripts for PLMVd and ESFY phytoplasma, 40 to 4 × 107 RNA transcripts for ACLSV, PPV and PDV, and 102 to 108 RNA transcripts for PNRSV, respectively. The duplex RT-qPCR assays, which were validated using both characterized isolates from all pathogens and field samples from Prunus species in Northern Greece, exhibited a broad detection range. Overall, the developed methods comprise useful tools that could be applied for the simultaneous and reliable detection of graft-transmissible pathogens in certification programs of Prunus spp.

Detection of 16SrVI and 16SrIX phytoplasma groups in pot marigold and tickseed plants in northeastern Iran.

Pot marigold and tickseed are ornamental plants with many medicinal and cosmetic uses and for landscape, respectively. During a survey in 2018, phyllody symptoms were observed in high percentages in these plants in some regions of the Razavi Khorasan province (northeastern Iran). Total DNA was extracted from symptomatic and asymptomatic plants and polymerase chain reaction was carried on using universal phytoplasma primer pairs P1/P7 and nested primer pairs R16F2n/R16R2. The nested amplification of 1200-bp fragments confirmed the presence of phytoplasmas only in the symptomatic plants. BLAST search, phylogenetic analysis, and virtual RFLP patterns of cloned amplicons allowed to classify the pot marigold phyllody phytoplasma in the 16SrVI-A subgroup while tickseed phyllody was enclosed in the 16SrIX-I subgroup. This is the first report of the association of a 16SrVI phytoplasma with pot marigold phyllody in Iran and of the phytoplasma presence in tickseed.

Widespread occurrence of 'Candidatus Phytoplasma ulmi' in elm species in Germany.

BACKGROUND: 'Candidatus Phytoplasma ulmi' is the agent associated with elm yellows and has been categorised in the European Union as a quarantine pathogen. For central and northern European countries, information on the occurrence and distribution of the pathogen and its impact on elms is scarce, so a survey of native elm trees has been conducted in Germany. RESULTS: About 6500 samples from Ulmus minor, Ulmus laevis and Ulmus glabra, were collected nationwide. Phytoplasma detection was performed by applying a universal 16Sr DNA-based quantitative PCR (qPCR) assay and a novel 'Ca. P. ulmi' specific qPCR assay targeting the 16S-23S spacer region. Both assays revealed that 28% of the samples were infected by 'Ca. P. ulmi', but infection rates of the elm species and regional incidences differed. The phytoplasma presence in the trees was not correlated to disease-specific symptoms. The survey identified a regional disparity of infection which was high in east, south and central Germany, whereas only a few infected sites were found in the western and northern parts of the country. Monitoring the seasonal titre of 'Ca. P. ulmi' in an infected tree by qPCR revealed a high colonisation in all parts of the tree throughout the year. CONCLUSIONS: 'Ca. P. ulmi' is widely present in elms in Germany. The rare occurrence of symptoms indicates either a high degree of tolerance in elm populations or a low virulence of pathogen strains enabling high infection rates in a long-living host.

When a Palearctic bacterium meets a Nearctic insect vector: Genetic and ecological insights into the emergence of the grapevine Flavescence dorée epidemics in Europe.

Flavescence dorée (FD) is a European quarantine grapevine disease transmitted by the Deltocephalinae leafhopper Scaphoideus titanus. Whereas this vector had been introduced from North America, the possible European origin of FD phytoplasma needed to be challenged and correlated with ecological and genetic drivers of FD emergence. For that purpose, a survey of genetic diversity of these phytoplasmas in grapevines, S. titanus, black alders, alder leafhoppers and clematis were conducted in five European countries. Out of 132 map genotypes, only 11 were associated to FD outbreaks, three were detected in clematis, whereas 127 were detected in alder trees, alder leafhoppers or in grapevines out of FD outbreaks. Most of the alder trees were found infected, including 8% with FD genotypes M6, M38 and M50, also present in alders neighboring FD-free vineyards and vineyard-free areas. The Macropsinae Oncopsis alni could transmit genotypes unable to achieve transmission by S. titanus, while the Deltocephalinae Allygus spp. and Orientus ishidae transmitted M38 and M50 that proved to be compatible with S. titanus. Variability of vmpA and vmpB adhesin-like genes clearly discriminated 3 genetic clusters. Cluster Vmp-I grouped genotypes only transmitted by O. alni, while clusters Vmp-II and -III grouped genotypes transmitted by Deltocephalinae leafhoppers. Interestingly, adhesin repeated domains evolved independently in cluster Vmp-I, whereas in clusters Vmp-II and-III showed recent duplications. Latex beads coated with various ratio of VmpA of clusters II and I, showed that cluster II VmpA promoted enhanced adhesion to the Deltocephalinae Euscelidius variegatus epithelial cells and were better retained in both E. variegatus and S. titanus midguts. Our data demonstrate that most FD phytoplasmas are endemic to European alders. Their emergence as grapevine epidemic pathogens appeared restricted to some genetic variants pre-existing in alders, whose compatibility to S. titanus correlates with different vmp gene sequences and VmpA binding properties.

Phytoplasmas diversity and identification of new aster yellows subgroup (16SrI) associated with weed species in Argentina.

Symptoms of phytoplasma infection were observed in different weed species, Bidens subalternans, Conyza bonariensis, Heterosperma ovatifolium and Conium maculatum, collected from diverse geographical regions in Argentina. To confirm the association of phytoplasma infection with symptomatic plants, PCR, RFLP and phylogenetic analyses based on 16S rRNA-encoding sequences were performed. In this work, we report the presence of phytoplasmas from group 16SrVII (subgroup 16VII-B) infecting C. bonariensis and B. subalternans and from group 16SrIII (subgroup 16SrIII-X) B. subalternans, H. ovatifolium, and C. maculatum. Phytoplasmas from the aster yellows group were detected infecting C. bonariensis and B. subalternans. Analysis of 16S rRNA-encoding genes revealed the presence of two distinct operons, rrnB (16SrI-B) and newly described rrnA, which is different from the reference RFLP patterns of all previously established 16SrI-subgroups. A single rp operon sequence analysis reveals the presence of simple infection and confirms a description of a novel subgroup. On the basis of these results we propose a designation of new subgroup 16SrI-(B/AJ) AJ (rp-AJ). To our knowledge, this is the first report of phytoplasmas infecting Bidens subalternans¸ Heterosperma ovatifolium and Conium maculatum.

Molecular and biological characterization of phytoplasmas from coconut palms affected by the lethal yellowing disease in Africa.

Côte d'Ivoire lethal yellowing (CILY) is a devastating disease associated with phytoplasmas and has recently rapidly spread to several coconut-growing areas in the Country. Phytoplasmas are phloem-restricted bacteria that affect plant species worldwide. These bacteria are transmitted by plant sap-feeding insects, and their cultivation was recently achieved in complex artificial media. In this study, phytoplasmas were isolated for the first time from coconut palm trunk borings in both solid and liquid media from CILY symptom-bearing and symptomless coconut palms. The colony morphology, PCR and sequencing analyses indicated the presence of phytoplasmas from different ribosomal groups. This study reports the first biochemical characterization of two of these phytoplasma isolates. Moreover, a disc-diffusion antibiotic susceptibility assay revealed that these bacteria exhibit tobramycin susceptibility and cephalexin hydrate and rifampicin resistance. Urea and arginine hydrolysis, and glucose fermentation tests that were performed on colonies of phytoplasmas and Acholeplasma laidlawii indicated that both phytoplasmas tested were negative for urea and positive for glucose and arginine, whereas A. laidlawii was positive for glucose and negative for urea and arginine. The growth of coconut phytoplasmas in both solid and liquid artificial media and the biological characterization of these isolates are novel and important advancements in the field of disease management and containment measures for the CILY disease. The characterization of isolated phytoplasmas will allow for more efficient management strategies in both the prevention of a coconut phytoplasma epidemics and the reduction of the economic impact of the disease in the affected areas.

Multilocus Sequence Analysis Reveals Three Distinct Populations of "Candidatus Phytoplasma palmicola" with a Specific Geographical Distribution on the African Continent.

To sustain epidemiological studies on coconut lethal yellowing disease (CLYD), a devastating disease in Africa caused by a phytoplasma, we developed a multilocus sequence typing (MLST) scheme for "Candidatus Phytoplasma palmicola" based on eight housekeeping genes. At the continental level, eight different sequence types were identified among 132 "Candidatus Phytoplasma palmicola"-infected coconuts collected in Ghana, Nigeria, and Mozambique, where CLYD epidemics are still very active. "Candidatus Phytoplasma palmicola" appeared to be a bacterium that is subject to strong bottlenecks, reducing the fixation of positively selected beneficial mutations into the bacterial population. This phenomenon, as well as a limited plant host range, might explain the observed country-specific distribution of the eight haplotypes. As an alternative means to increase fitness, bacteria can also undergo genetic exchange; however, no evidence for such recombination events was found for "Candidatus Phytoplasma palmicola." The implications for CLYD epidemiology and prophylactic control are discussed. The usefulness of seven housekeeping genes to investigate the genetic diversity in the genus "Candidatus Phytoplasma" is underlined.IMPORTANCE Coconut is an important crop for both industry and small stakeholders in many intertropical countries. Phytoplasma-associated lethal yellowing-like diseases have become one of the major pests that limit coconut cultivation as they have emerged in different parts of the world. We developed a multilocus sequence typing scheme (MLST) for tracking epidemics of "Ca Phytoplasma palmicola," which is responsible for coconut lethal yellowing disease (CLYD) on the African continent. MLST analysis applied to diseased coconut samples collected in western and eastern African countries also showed the existence of three distinct populations of "Ca Phytoplasma palmicola" with low intrapopulation diversity. The reasons for the observed strong geographic patterns remain to be established but could result from the lethality of CLYD and the dominance of short-distance insect-mediated transmission.

Phytoplasmas: An Introduction.

Phytoplasmas are among the most recently discovered plant pathogens. They are wall-less prokaryotes restricted to phloem tissue, associated with diseases affecting several hundred plant species. The impact of phytoplasma diseases on agriculture is impressive and, at the present day, no effective curative strategy has been developed. The availability of rapid and sensitive techniques for phytoplasma detection as well as the possibility to study their relationship with the host plants is a prerequisite for the management of phytoplasma-associated diseases.

Sampling Methods for Leafhopper, Planthopper, and Psyllid Vectors.

To reduce the spread of phytoplasmas in a crop or in a certain geographic area, epidemiological studies are of crucial importance in determining which insect species transmit these pathogens. In this chapter, we describe methods of capturing the insect vectors of phytoplasmas and the criteria for choosing the method(s) according to the objective to be achieved.

Comparison of Different Procedures for DNA Extraction for Routine Diagnosis of Phytoplasmas.

This chapter presents five different procedures for extracting DNA from phytoplasma-infected plants and insect vectors suitable for PCR assays. One of these procedures enriches phytoplasma DNA through differential centrifugation and is effective in producing highly purified DNA from fresh tissues from a wide variety of herbaceous and woody plants. Although the DNA yield is less than those of other known total DNA extraction procedures, a major advantage of the presented phytoplasma-enriched procedure is that a substantial proportion of the isolated DNA is from phytoplasmas. The other four procedures here presented involve treatments with CTAB-based buffer to lyse cells and purify DNA followed by deproteination and recovery of DNA. These procedures work well for extracting total DNA from fresh, frozen, or lyophilized tissues from a wide variety of plant hosts as well as insect vectors. Because few manipulations are required, the CTAB-based procedures are faster and easier to perform than the phytoplasma-enrichment protocol. In addition, they result in very high yields and provide DNA that is less pure but of suitable quality for the use in standard molecular biological techniques including PCR assays.

Standard Detection Protocol: PCR and RFLP Analyses Based on 16S rRNA Gene.

Phytoplasma detection and identification is primarily based on PCR followed by restriction fragment length polymorphism analysis. This method detects and differentiates phytoplasmas including those not yet identified. The protocol describes the application of this method for identification of phytoplasmas at 16S rRNA (16Sr) group and 16Sr subgroup levels on amplicons and also in silico on the same sequences.