Phytoplasma and phytoplasma diseases: a review of recent research

Numerous yellows-type diseases of plants have been associated with wall-less prokaryote pathogens \u2013 phytoplasma over the last 40 years. These pathogens cannot be grown in axenic culture so that advances in their study are mainly achieved by molecular techniques. Severe disease epidemics associated with a phytoplasma presence have been described worldwide. These include coconut lethal yellowing in Africa and the Caribbean, grapevine yellows in major viticultural areas and various diseases affecting stone and pome fruit plants. Phytoplasma-infected plants exhibit symptoms suggesting a profound disturbance in the normal balance of growth regulators and also yellows symptoms, but very often the symptomatology is not diagnostic. Detection and characterization of phytoplasmas infecting different plant species are now possible with molecular methods, based on the study of 16S rDNA polymorphisms. Molecular diversity of phytoplasmas is also demonstrated by studying genes coding the ribosomal proteins S3, tuf, SecY, amp, imp and other genes. Four phytoplasma genomes have been fully sequenced, including those of two \u2018Candidatus Phytoplasma asteris\u2019 strains, and those of strains of \u2018Ca. P. mali\u2019 and \u2018Ca. P. australiense\u2019. Three of these genomes contain large amounts of repeated DNA sequence, and the fourth carries multiple copies of almost 100 genes. Considering that phytoplasmas have unusually small genomes, these repeats might be related to their transkingdom habitat and to their pathogenic activity. An outlook of recent findings in the field is also repor

Detection and identification of phytoplasmas in two vineyards located in a restricted geographic area in Italy

A survey to verify the presence of grapevine yellows was carried out in a restricted area of the Marche region (central east Italy) in a small geographic area. In two locations six grapevine varieties were examined and phytoplasmas belonging to four ribosomal groups were detected by PCR/RFLP analyses. The surveyed varieties were Passerina, Pecorino, Merlot, Sangiovese, Fedit and Montepulciano. The different phytoplasmas detected were present in a scattered manner in all the varieties, but only Passerina, Pecorino and Sangiovese where shown to be infected by “bois noir” (16SrXII-A) phytoplasmas. Considering the landscape shape and the age of the vineyards the source of BN infection and of the other phytoplasmas is very likely in the surrounding environment or vineyards

Isolation of diverse phytoplasmas from symptomatic grapevine samples

Phytoplasmas from different ribosomal groups were isolated from grapevine samples in which “flavescence dorée” or “bois noir” phytoplasmas had been identified. The results indicate that the developed medium is not phytoplasma specific and supports the growth of phytoplasmas which cannot be detected by other molecular methods and are very likely present in a very low titre in the endobiome of the plants. The applied method discriminates the presence of bacterial contamination already in the liquid phase, and the colony purification technique allows excluding the contamination

Papers Ribosomal protein coding genes SSU12p and LSU36p as molecular markers for phytoplasma detection and differentiation

Detection and classification of phytoplasmas mainly rely on amplification of the 16S rRNA gene followed by RFLP analysis and/or sequencing, because these organisms lack complete phenotypic characterization. Other conserved genomic loci have been exploited as additional molecular markers for phytoplasma differentiation. Two loci, SSU12p and LSU36p, selected by whole-genome comparison of 12 phytoplasma strains, were used for primer design, and were successfully tested on DNA samples from plants infected by phytoplasmas belonging to ten 16S ribosomal groups. The phylogenetic trees inferred from SSU12p and LSU36p loci were highly congruent to the trees derived from 16S rRNA and tuf genes of the same phytoplasma strains. Virtual RFLP analysis of the amplified SSU12p gene showed distinct patterns for most of the phytoplasma ribosomal subgroups tested. These results show that SSU12p and LSU36p genes are reliable additional markers for phytoplasma detection and differentiation

Identification of host genes potentially implicated in the Malus pumila and ‘Candidatus Phytoplasma mali’ interactions

Two‘Candidatus Phytoplasma mali’ strains (AP and AT), were studied in experimentally infected apple trees to analyze transcriptional profiles during interaction with phytoplasmas. Three groups of sample combinations were employed: healthy - infected, symptomatic - non-symptomatic, and AP-infected - AT-infected sample. The majority of genes were differently expressed between healthy and infected samples. Changes in gene expression involved a wide spectrum of biological functions, including processes of metabolism, cell defence, photosynthesis, transport, transcription, signal transduction and protein synthesis. The possible effect of phytoplasma infection on these processes and their relationships with disease development, symptom appearance and possible plant defence system is discussed. Keywords: Apple, phytoplasmas, ‘Ca. P. mali’, gene expression, transcriptom

Occurrence and molecular characterization of a 16SrI-R subgroup phytoplasma associated with Aquilegia vulgaris phyllody disease

During 2016–2017 surveys, carried out for phytoplasma diseases in ornamental plants in Chaharmahal and Bakhtiari provinces, Iran, found symptoms of virescence, phyllody, reduced size of leaves and flowers in columbine (Aquilegia vulgaris). Total DNAs extracted from symptomatic and symptomless plants were tested for the presence of phytoplasma using P1/P7 and R16F2n/R16R2 primers in direct and nested PCR producing amplicons of about 1.8 and 1.2 kb, respectively, from all symptomatic A. vulgaris plants, but not from symptomless ones. The consensus sequence of the detected phytoplasma named Aquilegia phyllody (APh) was 100% identical with strains clustering to phytoplasmas enclosed in the 16SrI group as also confirmed by phylogenetic analyses. Both real and virtual restriction fragment length polymorphism analysis of R16F2n/R16R2 amplicons showed profiles that were identical to each other and indicated the affiliation of the APh phytoplasma to the 16SrI-R subgroup. This is the first report of a 16SrI-R phytoplasma associated with this A. vulgaris phyllody disease

Assessment of susceptibility to European stone fruit yellows phytoplasma of new plum variety and five rootstock/plum variety combinations

Two separate experiments were carried out to assess the plum susceptibility to infection by European stone fruit yellows phytoplasmas during a five years period. Commercial varieties/cultivars and new selections grafted on Myrabolan 29C were evaluated in at least two plots of four plants each. Visual inspection and PCR/RFLP identification of phytoplasmas detected an increasing phytoplasma presence in both symptomatic and asymptomatic plants. Eight Japanese plum selections showed ESFY symptoms or pathogen presence in the 50% of the plants and nine selections showed ESFY infection in 20% of the plants. Only nine selections showed absence of both symptoms and pathogen. Although the European selections/cultivars were not symptomatic, plants belonging to six of these cultivars were positive for phytoplasma infection. The evaluation of cultivar/rootstock combinations indicate phytoplasma presence from the first year after plantation on. Two of the rootstocks seem to induce a delay in symptoms appearance and cultivar T.C. Sun resulted to be the most susceptible to the disease independently from the rootstock employed. Keywords: Japanese plum, European plum, European stone fruit yellows phytoplasmas, resistance, disease

Grapevine yellows diseases in Spain: eight years survey of disease spread and molecular characterization of phytoplasmas involved.

Among grapevine yellows phytoplasma diseases in Europe, flavescence dor\ue9e (FD) is the most devastating and in the last decade has reached Spanish vineyards, mainly in Catalonia. An eight-year survey was carried out in the areas where the disease has spread (Alt Empord\ue0, Catalonia, Northern Spain) and in the remaining vine-growing areas of Catalonia. Sequence analyses of a portion of the 16S-23S ribosomal DNA cistron, from selected grapevine samples from Catalonia, showed that the phytoplasmas involved in grapevine yellows belong to 16S ribosomal subgroups V-D (flavescence dor\ue9e, FD) and XII-A (bois noir, BN). A set of Spanish FD isolates collected during these years were further studied by RFLP analyses of the 16S-23S ribosomal DNA fragment, as well as the rpS3 and SecY genes. All the FD phytoplasma strains studied were related to phytoplasmas belonging to ribosomal protein subgroup rp-E

Molecular variability and host distribution of ‘candidatus phytoplasma solani’ strains from different geographic origins

The knowledge of phytoplasma genetic variability is a tool to study their epidemiology and to implement an effective monitoring and management of their associated diseases. ‘Candidatus Phytoplasma solani’ is associated with “bois noir” disease in grapevines, and yellowing and decline symptoms in many plant species, causing serious damages during the epidemic outbreaks. The epidemiology of the diseases associated with this phytoplasma is complex and related to numerous factors, such as interactions of the host plant and insect vectors and spreading through infected plant propagation material. The genetic variability of ‘Ca. P. solani’ strains in different host species and in different geographic areas during the last two decades was studied by RFLP analyses coupled with sequencing on vmp1, stamp, and tuf genes. A total of 119 strains were examined, 25 molecular variants were identified, and the variability of the studied genes was linked to both geographic distribution and year of infection. The crucial question in ‘Ca. P. solani’ epidemiology is to trace back the epidemic cycle of the infections. This study presents some relevant features about differential strain distribution useful for disease monitoring and forecasting, illustrating and comparing the phytoplasma molecular variants identified in various regions, host species, and time periods

Experimental transmission by Scaphoideus titanus Ball of two Flavescence doree-type phytoplasmas

During the last 5 years Flavescence doree (FD) has been expanding from the first loci of outbreak in the Veneto region, to the major viticultural areas of northern Italy, showing increased infection rates in 1999. The FD vector is Scaphoideus titanus Ball. Recently it has been demonstrated that in Veneto FD is associated with two molecularly distinguishable types of phytoplasmas, both belonging to the 16SrV group (Elm yellows and related strains). In 1997 the two phytoplasma types appeared to be geographically separated in 1997: FD-16SrV-D was found in the provinces of Verona, Vicenza, Venice and Padova, infecting mainly cvs Garganega and Chardonnay while FD-16SrV-C occurred in the Treviso province, infecting cultivars Prosecco, Chardonnay and Perera. During 1998 the two types started to merge in Treviso province, showing that 16SrV-D was transmitted more efficently and over longer distances than the other type. From the transmission trials carried out in 1998 and 1999 in order to gain more information about the epidemiology of the two types of FD agents, S. titanus was found to be able to transmit both 16SrV-C and 16SrV-D phytoplasmas.