Almond witches’-broom (AlmWB), a devastating disease of almond, peach and nectarine in Lebanon, is associated with ‘Candidatus Phytoplasma phoenicium’. In the present study, we generated a draft genome sequence of ‘Ca. P. phoenicium’ strain SA213, representative of phytoplasma strain populations from different host plants, and determined the genetic diversity among phytoplasma strain populations by phylogenetic analyses of 16S rRNA, groEL, tufB and inmp gene sequences. Sequence-based typing and phylogenetic analysis of the gene inmp, coding an integral membrane protein, distinguished AlmWB-associated phytoplasma strains originating from diverse host plants, whereas their 16S rRNA, tufB and groEL genes shared 100 % sequence identity. Moreover, dN/dS analysis indicated positive selection acting on inmp gene. Additionally, the analysis of ‘Ca. P. phoenicium’ draft genome revealed the presence of integral membrane proteins and effector-like proteins and potential candidates for interaction with hosts. One of the integral membrane proteins was predicted as BI-1, an inhibitor of apoptosis-promoting Bax factor. Bioinformatics analyses revealed the presence of putative BI-1 in draft and complete genomes of other ‘Ca. Phytoplasma’ species. The genetic diversity within ‘Ca. P. phoenicium’ strain populations in Lebanon suggested that AlmWB disease could be associated with phytoplasma strains derived from the adaptation of an original strain to diverse hosts. Moreover, the identification of a putative inhibitor of apoptosis-promoting Bax factor (BI-1) in ‘Ca. P. phoenicium’ draft genome and within genomes of other ‘Ca. Phytoplasma’ species suggested its potential role as a phytoplasma fitness-increasing factor by modification of the host-defense response.

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‘Candidatus Phytoplasma phoenicium’ associated with almond witches’-broom disease: from draft genome to genetic diversity among strain populations

Several economically relevant phytoplasma-associated diseases are described together with an update of phytoplasma taxonomy and major biological and molecular features of phytoplasmas. Outlook about persepectives and future work to contain spread of these diseases are also reported.

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Phytoplasmas and Phytoplasma Diseases: A Severe Threat to Agriculture

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.

Phytoplasmas: diversity, taxonomy, and epidemiology.

Phytoplasmas classified in group 16SrXII infect a wide range of plants and are transmitted by polyphagous planthoppers of the family Cixiidae. Based on 16S rRNA gene sequence identity and biological properties, group 16SrXII encompasses several species, including 'Candidatus Phytoplasma australiense', 'Candidatus Phytoplasma japonicum' and 'Candidatus Phytoplasma fragariae'. Other group 16SrXII phytoplasma strains are associated with stolbur disease in wild and cultivated herbaceous and woody plants and with bois noir disease in grapevines (Vitis vinifera L.). Such latter strains have been informally proposed to represent a separate species, 'Candidatus Phytoplasma solani', but a formal description of this taxon has not previously been published. In the present work, stolbur disease strain STOL11 (STOL) was distinguished from reference strains of previously described species of the 'Candidatus Phytoplasma' genus based on 16S rRNA gene sequence similarity and a unique signature sequence in the 16S rRNA gene. Other stolbur- and bois noir-associated ('Ca. Phytoplasma solani') strains shared >99 % 16S rRNA gene sequence similarity with strain STOL11 and contained the signature sequence. 'Ca. Phytoplasma solani' is the only phytoplasma known to be transmitted by Hyalesthes obsoletus. Insect vectorship and molecular characteristics are consistent with the concept that diverse 'Ca. Phytoplasma solani' strains share common properties and represent an ecologically distinct gene pool. Phylogenetic analyses of 16S rRNA, tuf, secY and rplV-rpsC gene sequences supported this view and yielded congruent trees in which 'Ca. Phytoplasma solani' strains formed, within the group 16SrXII clade, a monophyletic subclade that was most closely related to, but distinct from, that of 'Ca. Phytoplasma australiense'-related strains. Based on distinct molecular and biological properties, stolbur- and bois noir-associated strains are proposed to represent a novel species level taxon, 'Ca. Phytoplasma solani'; STOL11 is designated the reference strain.

'Candidatus Phytoplasma solani', a novel taxon associated with stolbur- and bois noir-related diseases of plants.

A new real-time PCR detection system was developed for grapevine yellows (GY) using TaqMan minor groove binder probes and including two amplicons for group-specific detection of Flavescence doree (FD) and Bois noir (BN) phytoplasmas, plus a universal phytoplasma amplicon. FD and BN amplicons were designed to amplify species-specific genomic DNA fragments and the universal amplicon to amplify the 16S ribosomal DNA region. Efficiency of PCR amplification, limit of detection, range of linearity and dynamic range were assessed for all three amplicons. The specificity of detection systems was tested on several other isolates of phytoplasmas and bacteria and on healthy field grapevine and insect samples. No cross-reactivity with other phytoplasma strains, plant or insect DNA was detected. The assay was compared with conventional PCR on more than 150 field grapevine, insect and field bindweed samples. Real-time PCR showed higher sensitivity as phytoplasmas were detected in several PCR-negative and in all PCR-positive samples. A data-mining analysis of results from both detection approaches also favoured real-time PCR over conventional PCR diagnostics. The developed procedure for detection of phytoplasmas in grapevine also included amplification of plant DNA co-extracted with phytoplasmic DNA, providing additional quality control for the DNA extraction and PCR amplification for each sample. The newly developed assay is a reliable, specific and sensitive method easily applicable to high-throughput diagnosis of GY.

Real‐time PCR detection systems for Flavescence dorée and Bois noir phytoplasmas in grapevine: comparison with conventional PCR detection and application in diagnostics

Grapevines, alternative host plants, and vectors from different viticultural areas of Germany were surveyed for the presence of stolbur-group phytoplasmas that are associated with grapevine yellows. Isolates from these field samples and periwinkle isolates of stolbur were characterised by RFLP-analysis of non-ribosomal DNA fragments using a combination of 6 polymorphic primer-enzyme combinations. Three of the 5 different restriction profiles that could be distinguished were found in grapevine and other field samples of plants and insects. A previously unknown isolate was detected in grapevine, vectors and the newly identified herbaceous host Calystegia sepium . The further analysis of the phylogenetic relationship between the 5 RFLP groups revealed a close relationship between two of the isolates associated with grapevine yellows which were also found in the two Convolvulaceae Convolvulus arvensis and Calystegia sepium . The third isolate which was detected in grapevine and stinging nettle Urtica dioica is more closely related to a periwinkle isolate obtained from Lavandula officinalis than to the other two grapevine yellows isolates. Based on the data of the field survey a specific association of the three isolates to different alternative host plants is discussed.

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M. Langer

Papers

Molecular characterisation of grapevine yellows associated phytoplasmas of the stolbur-group based on RFLP-analysis of non-ribosomal DNA