Revision of the 'Candidatus Phytoplasma' species description guidelines.

The genus 'Candidatus Phytoplasma' was proposed to accommodate cell wall-less bacteria that are molecularly and biochemically incompletely characterized, and colonize plant phloem and insect vector tissues. This provisional classification is highly relevant due to its application in epidemiological and ecological studies, mainly aimed at keeping the severe phytoplasma plant diseases under control worldwide. Given the increasing discovery of molecular diversity within the genus 'Ca. Phytoplasma', the proposed guidelines were revised and clarified to accommodate those 'Ca. Phytoplasma' species strains sharing >98.65 % sequence identity of their full or nearly full 16S rRNA gene sequences, obtained with at least twofold coverage of the sequence, compared with those of the reference strain of such species. Strains sharing <98.65 % sequence identity with the reference strain but >98.65 % with other strain(s) within the same 'Ca. Phytoplasma' species should be considered related strains to that 'Ca. Phytoplasma' species. The guidelines herein, keep the original published reference strains. However, to improve 'Ca. Phytoplasma' species assignment, complementary strains are suggested as an alternative to the reference strains. This will be implemented when only a partial 16S rRNA gene and/or a few other genes have been sequenced, or the strain is no longer available for further molecular characterization. Lists of 'Ca. Phytoplasma' species and alternative reference strains described are reported. For new 'Ca. Phytoplasma' species that will be assigned with identity ≥98.65 % of their 16S rRNA gene sequences, a threshold of 95 % genome-wide average nucleotide identity is suggested. When the whole genome sequences are unavailable, two among conserved housekeeping genes could be used. There are 49 officially published 'Candidatus Phytoplasma' species, including 'Ca. P. cocostanzaniae' and 'Ca. P. palmae' described in this manuscript.

Recommended rejection of the names Malacoplasma gen. nov., Mesomycoplasma gen. nov., Metamycoplasma gen. nov., Metamycoplasmataceae fam. nov., Mycoplasmoidaceae fam. nov., Mycoplasmoidales ord. nov., Mycoplasmoides gen. nov., Mycoplasmopsis gen. nov. [Gupta, Sawnani, Adeolu, Alnajar and Oren 2018] and all proposed species comb. nov. placed therein.

The consensus of the members of the International Committee on Systematics of Prokaryotes' Subcommittee on the taxonomy of Mollicutes is that recently proposed sweeping changes to nomenclature of members of the Mycoplasmatales, specifically involving introduction of the names Malacoplasma gen. nov., Mesomycoplasma gen. nov., Metamycoplasma gen. nov., Metamycoplasmataceaefam. nov., Mycoplasmoidaceaefam. nov., Mycoplasmoidalesord. nov., Mycoplasmoides gen. nov., Mycoplasmopsis gen. nov., and all proposed species or subspecies comb. nov. placed therein, should be rejected because they violate one or more essential points of the International Code of Nomenclature of Prokaryotes.

Molecular Diversity of Phytoplasmas Associated with Grapevine Yellows Disease in North-Eastern Italy.

A 3-year survey was conducted in Northern Italy to verify the presence and diversity of phytoplasmas in selected vineyards showing symptoms of severe yellows. Symptomatic and asymptomatic grapevines were sampled, and insects were collected using yellow sticky traps. The phytoplasmas detected in grapevine samples were different according to the years: "flavescence dorée" (16SrV-C/D) was detected together with other phytoplasmas such as 16SrXII-A ('Candidatus Phytoplasma solani'-related, bois noir), 16SrI-B ('Ca. P. asteris'-related, aster yellows), 16SrX-B ('Ca. P. prunorum'-related, European stone fruit yellows), and 16SrV-A ('Ca. P. ulmi'-related, elm yellows). Moreover, phytoplasmas belonging to 16SrVII-A ('Ca. P. fraxini'-related) and 16SrVI ('Ca. P. trifolii'-related) subgroups were also identified. Identification of phytoplasmas was also carried out from insects and showed the presence of some of these phytoplasmas in Scaphoideus titanus and Orientus ishidae: 16SrXII-A, 16SrVII, and 16SrVI phytoplasmas were detected in specimens of both species, while 16SrXII-A and 16SrI-B phytoplasma strains were identified in Orientus ishidae and Hyalesthes obsoletus, and 16SrX-B in S. titanus. Direct sequencing of selected amplicons obtained from 16S rRNA, rp, and tuf genes from grapevine and insect samples confirmed the phytoplasma identification. The 16SrVII-A and 16SrVI phytoplasmas were never detected before in grapevine, S. titanus and Orientus ishidae in Europe and their epidemiological importance is being monitored.

Draft Whole Genome Sequence Analyses on Pseudomonas syringae pv. actinidiae Hypersensitive Response Negative Strains Detected from Kiwifruit Bleeding Sap Samples.

Kiwifruit bleeding sap samples, collected in Italian and Chilean orchards from symptomatic and asymptomatic plants, were evaluated for the presence of Pseudomonas syringae pv. actinidiae, the causal agent of bacterial canker. The saps were sampled during the spring in both hemispheres, before the bud sprouting, during the optimal time window for the collection of an adequate volume of sample for the early detection of the pathogen, preliminarily by molecular assays, and then through its direct isolation and identification. The results of molecular analyses showed more effectiveness in the P. syringae pv. actinidiae detection when compared with those of microbiological analyses through the pathogen isolation on the nutritive and semiselective media selected. The bleeding sap analyses allowed the isolation and identification of two hypersensitive response (HR) negative and hypovirulent P. syringae pv. actinidiae strains from different regions in Italy. Moreover, multilocus sequence analysis (MLSA) and whole genome sequence (WGS) were carried out on selected Italian and Chilean P. syringae pv. actinidiae virulent strains to verify the presence of genetic variability compared with the HR negative strains and to compare the variability of selected gene clusters between strains isolated in both countries. All the strains showed the lack of argK and coronatine gene clusters as reported for the biovar 3 P. syringae pv. actinidiae strains. Despite the biologic differences obtained in the tobacco bioassays and in pathogenicity assays, the MLSA and WGS analyses did not show significant differences between the WGS of the HR negative and HR positive strains; the difference, on the other hand, between PAC_ICE sequences of Italian and Chilean P. syringae pv. actinidiae strains was confirmed. The inability of the hypovirulent strains IPV-BO 8893 and IPV-BO 9286 to provoke HR in tobacco and the low virulence shown in this host could not be associated with mutations or recombinations in T3SS island.

General phytoplasma detection by a q-PCR method using mycoplasma primers.

Phytoplasmas and mycoplasmas are bacteria belonging to the class Mollicutes. In this study, a fine tuning of quantitative polymerase chain reaction (qPCR) with a universal mycoplasma primer pair (GPO3F/MGSO) targeting the 16S rRNA gene was carried out on phytoplasmas. The dissociation curves of DNAs from Catharanthus roseus phytoplasma-infected micropropagated shoots and from phytoplasma field-infected plant samples showed a single peak at 82.5 °C (±0.5) specifically detecting phytoplasmas belonging to several ribosomal groups. Assay specificity was determined with DNA of selected bacteria: 'Candidatus Liberibacter solanacearum', Xylella fastidiosa, Ralstonia solanacearum and Clavibacter michiganensis. No amplification curves were observed with any of these tested bacteria except 'Ca. L. solanacearum' that was amplified with a melting temperature at 85 °C. Absolute quantification of phytoplasma titer was calculated using standard curves prepared from serial dilutions of plasmids containing the cloned fragment GPO3F/MGSO from European stone fruit yellows phytoplasma. Phytoplasma copy number ranged from 106 to 103 according with the sample. The sensitivity evaluated comparing plasmid serial dilutions resulted 10-6 for conventional PCR and 10-7 for qPCR. The latter method resulted therefore able to detect very low concentrations of phytoplasma in plant material.

Potential Applications and Limitations of Electronic Nose Devices for Plant Disease Diagnosis.

Electronic nose technology has recently been applied to the detection of several plant diseases and pests, with promising results. However, in spite of its numerous advantages, including operational simplicity, non-destructivity, and bulk sampling, drawbacks include a low sensitivity and specificity in comparison with microbiological and molecular methods. A critical review of the use of an electronic nose for plant disease diagnosis and pest detection is presented, describing the instrumental and procedural advances of sensorial analysis, for the improvement of discrimination between healthy and infected or infested plants. In conclusion, the use of electronic nose technology is suggested to assist, direct, and optimise traditionally adopted diagnostic techniques.

Multigene characterization of a new 'Candidatus Phytoplasma rubi'-related strain associated with blackberry witches' broom.

A new phytoplasma was identified in naturally infected blackberry plants exhibiting witches' broom symptoms in Portugal. The 16S rRNA gene sequence revealed that it is related to 'Candidatus Phytoplasma rubi' (16SrV-E ribosomal subgroup) and RFLP analysis revealed a unique profile following MseI endonuclease digestion of R16F2n/R2 amplicons that distinguished it from the strains belonging to previously established 16SrV phytoplasma subgroups. The in silico restriction analyses confirmed that the phytoplasma strain from blackberry is different from all the other strains reported in group 16SrV. Phylogeny of the 16S rRNA gene sequences, sequence analyses of 16S-23S, tuf, rplV-rpsC, rplF-rplR, rplO-SecY-map and uvrB-degV genetic loci, as well as the variability of unique oligonucleotide sequences defined for 'Candidatus Phytoplasma rubi' confirmed the uniqueness of this phytoplasma strain from Portugal for which a novel ribosomal subgroup, 16SrV-I, is proposed. The representative of this new subgroup was named blackPort phytoplasma (Portuguese blackberry phytoplasma).

Differentiation of 'Candidatus Phytoplasma cynodontis' Based on 16S rRNA and groEL Genes and Identification of a New Subgroup, 16SrXIV-C.

'Candidatus Phytoplasma cynodontis' is widespread in bermudagrass and has only been found in monocotyledonous plants. Molecular studies carried out on strains collected in Italy, Serbia, and Albania enabled verification of molecular variability in the 16S ribosomal RNA (rRNA) gene. Based on restriction fragment length polymorphism and sequence analyses, the strains from Serbia were clearly differentiated from all others and assigned to a new ribosomal DNA (rDNA) subgroup designated as 16SrXIV-C. A system for amplification of fragments containing the 'Ca. P. cynodontis' groEL gene was developed to enable study of its variability in related strains belonging to different 16SrXIV subgroups. Despite the fact that the groEL gene exhibited a greater sequence variation than 16S rRNA, the phylogenetic tree based on groEL gene sequence analysis was highly congruent with the 16S rDNA-based tree. The groEL gene analyses supported differentiation of the Serbian strains and definition of the new subgroup 16SrXIV-C. Phylogenetic analyses of both genes confirmed distinct phylogenetic lineages for strains belonging to 16SrXIV subgroups. Furthermore, groEL is the only nonribosomal marker developed for characterization of 'Ca. P. cynodontis' thus far, and its application in molecular surveys should provide better insight into the relationships among these phytoplasmas and correlation between strain differentiation and their geographical distribution.

'Candidatus Phytoplasma asteris' Strains Associated with Oil Palm Lethal Wilt in Colombia.

The distribution of lethal wilt, a severe disease of oil palm, is spreading throughout South America. An incidence of about 30% was recorded in four commercial fields in Colombia. In this study, phytoplasmas were detected in symptomatic oil palm by using specific primers, based on 16S ribosomal DNA (rDNA) sequences, in nested polymerase chain reaction assays. The phytoplasmas were then identified as 'Candidatus Phytoplasma asteris', ribosomal subgroup 16SrI-B, through the use of restriction fragment length polymorphism (RFLP) analysis and sequencing. Cloning and sequencing of 16S rDNA from selected strains, together with phylogenetic analysis, confirmed the classification. Moreover, collective RFLP characterization of the groEL, amp, and rp genes, together with sequence data, distinguished the aster yellows strain detected in Colombian oil-palm samples from other aster yellows phytoplasmas used as reference strains; in particular, from an aster yellows strain infecting corn in the same country.

Phytoplasma-Associated Diseases in South America: Thirty Years of Research.

Phytoplasma-associated diseases are mainly insect-transmitted and are present worldwide. Considering that disease detection is a relevant environmental factor that may elucidate the presence of these diseases, a review reporting the geographic distribution of phytoplasma taxa in geographically consistent areas helps manage diseases appropriately and reduce their spreading. This work summarizes the data available about the identification of the phytoplasma associated with several diverse diseases in South America in the last decades. The insect vectors and putative vectors together with the plant host range of these phytoplasmas are also summarized. Overall, 16 'Candidatus Phytoplasma' species were detected, and those most frequently detected in agricultural-relevant crops such as corn, alfalfa, grapevine, and other horticultural species are 'Ca. P. pruni', 'Ca. P. asteris', and 'Ca. P. fraxini'.

Paulownia Witches' Broom Disease: A Comprehensive Review.

Phytoplasmas are insect-transmitted bacterial pathogens associated with diseases in a wide range of host plants, resulting in significant economic and ecological losses. Perennial deciduous trees in the genus Paulownia are widely planted for wood harvesting and ornamental purposes. Paulownia witches' broom (PaWB) disease, associated with a 16SrI-D subgroup phytoplasma, is a destructive disease of paulownia in East Asia. The PaWB phytoplasmas are mainly transmitted by insect vectors in the Pentatomidae (stink bugs), Miridae (mirid bugs) and Cicadellidae (leafhoppers) families. Diseased trees show typical symptoms, such as branch and shoot proliferation, which together are referred to as witches' broom. The phytoplasma presence affects the physiological and anatomical structures of paulownia. Gene expression in paulownia responding to phytoplasma presence have been studied at the transcriptional, post-transcriptional, translational and post-translational levels by high throughput sequencing techniques. A PaWB pathogenic mechanism frame diagram on molecular level is summarized. Studies on the interactions among the phytoplasma, the insect vectors and the plant host, including the mechanisms underlying how paulownia effectors modify processes of gene expression, will lead to a deeper understanding of the pathogenic mechanisms and to the development of efficient control measures.

Multilocus Gene Analyses Indicate Tamarix aphylla as Reservoir Host of Diverse Phytoplasmas Associated with Witches' Broom and Yellowing Symptomatology.

Tamarisk witches' broom, yellowing, and little leaf symptoms were observed during 2018-2023 surveys of rural deserts in central regions of Iran with the highest disease incidence up to 72% in Chah Afzal (Yazd province). A verification of the presence and identity of phytoplasmas associated with these symptoms was then performed. Tamarisk tree branch cuttings obtained from symptomatic plants sprouted up to 90.3% but with 15-25 days' delay compared to the asymptomatic ones and showed internode shortening and witches' broom, while the branch cuttings from asymptomatic plants had normal growth and sprouted up to 97.8%. Phytoplasma transmission by dodder bridges to periwinkle did not succeed, while nested polymerase chain reaction on the phytoplasma ribosomal gene followed by RFLP and phylogenetic analyses revealed the presence of 'Candidatus Phytoplasma asteris', 'Ca. P. australasiae=australasiaticum', and 'Ca. P. trifolii' (ribosomal subgroups 16SrI-B, 16SrII-D, and 16SrVI-A, respectively) in the samples from symptomatic plants only. Further amplifications were performed on selected phytoplasma-positive samples on tuf and secA genes, and the produced sequences indicated the presence of mixed phytoplasma infection in some of the samples. In particular, in the tuf gene, a mixed infection of 'Ca. P. australasiae=australasiaticum' and 'Ca. P. trifolii' was detected, while in the secA gene, the presence of 'Ca. P. asteris' or 'Ca. P. tritici' strains was identified. The first-time detection of diverse phytoplasma strains in symptomatic T. aphylla suggests that this species represent a relevant source of infection for the agricultural crops and for landscape plants especially when temperature allows insect vector transmission, and therefore, it represents a risk in every environment especially in the frame of climatic changes.

'Candidatus Phytoplasma balanitae' associated with witches' broom disease of Balanites triflora.

A phytoplasma was identified in naturally infected wild Balanites triflora plants exhibiting typical witches' broom symptoms (Balanites witches' broom: BltWB) in Myanmar. The 16S rRNA gene sequence revealed that BltWB phytoplasma had the highest similarity to that of 'Candidatus Phytoplasma ziziphi' and it was also closely related to that of 'Candidatus Phytoplasma ulmi'. Phylogenetic analysis of the 16S rRNA gene sequences indicated that the BltWB phytoplasma clustered as a discrete subclade with Elm yellows phytoplasmas. RFLP analysis of the 16S rRNA gene including the 16S-23S spacer region differentiated the BltWB phytoplasma from 'Ca. P. ziziphi', 'Ca. P. ulmi' and 'Candidatus Phytoplasma trifolii'. Analysis of additional ribosomal protein (rp) and translocase protein (secY) gene sequences and phylogenetic analysis of BltWB showed that this phytoplasma was clearly distinguished from those of other 'Candidatus Phytoplasma' taxa. Taking into consideration the unique plant host and the restricted geographical occurrence in addition to the 16S rRNA gene sequence similarity, the BltWB phytoplasma is proposed to represent a novel taxon, 'Candidatus Phytoplasma balanitae'.

Phloem-Localized Insect-Transmitted Bacteria Associated with Plant Diseases.

In the last three decades, an increasing number of plant diseases associated with the presence of phloem-localized insect-transmitted bacteria have been observed around the world, causing serious economic losses [...].

Small RNA Profiling of Aster Yellows Phytoplasma-Infected Catharanthus roseus Plants Showing Different Symptoms.

Micropropagated Catharantus roseus plants infected with 'Candidatus Phytoplasma asteris' showed virescence symptoms, witches' broom symptoms, or became asymptomatic after their planting in pots. Nine plants were grouped into three categories according to these symptoms, which were then employed for investigation. The phytoplasma concentration, as determined by qPCR, correlated well with the severity of symptoms. To reveal the changes in the small RNA profiles in these plants, small RNA high-throughput sequencing (HTS) was carried out. The bioinformatics comparison of the micro (mi) RNA and small interfering (si) RNA profiles of the symptomatic and asymptomatic plants showed changes, which could be correlated to some of the observed symptoms. These results complement previous studies on phytoplasmas and serve as a starting point for small RNA-omic studies in phytoplasma research.

'Candidatus Phytoplasma convolvuli', a new phytoplasma taxon associated with bindweed yellows in four European countries.

Plants of Convolvulus arvensis exhibiting symptoms of undersized leaves, shoot proliferation and yellowing, collectively defined as bindweed yellows, were sampled in different regions of Europe and assessed for phytoplasma infection by PCR amplification using phytoplasma universal rRNA operon primer pairs. Positive results were obtained for all diseased plants. RFLP analysis of amplicons comprising the16S rRNA gene alone or the16S rRNA gene and 16-23S intergenic spacer region indicated that the detected phytoplasmas were distinguishable from all other previously described rRNA gene sequences. Analysis of 16S rRNA gene sequences derived from seven selected phytoplasma strains (BY-S57/11, BY-S62/11, BY-I1015, BY-I1016, BY-BH1, BY-BH2 and BY-G) showed that they were nearly identical (99.9-100% gene sequence similarity) but shared less than 97.5% similarity with comparable sequences of other phytoplasmas. Thus, BY phytoplasmas represent a new taxon whose closest relatives are stolbur phytoplasma strains and 'Candidatus Phytoplasma fragariae' with which they share 97.2% and 97.1% 16S rRNA gene sequence similarity, respectively. Phylogenetic analysis of 16S rRNA gene sequences confirmed that bindweed yellows phytoplasma strains collectively represent a distinct lineage within the phytoplasma clade and share a common ancestor with previously published or proposed 'Candidatus Phytoplasma' taxa within a major branch including aster yellows and stolbur phytoplasmas. On the basis of unique 16S rRNA gene sequences and biological properties that include a single host plant species and a geographical distribution limited to parts of Europe, the bindweed yellows (BY) phytoplasmas represent a coherent but discrete taxon, 'Candidatus Phytoplasma convolvuli', with strain BY-S57/11 (GenBank accession no. JN833705) as the reference strain.

Plants and Phytoplasmas: When Bacteria Modify Plants.

Plant pathogen presence is very dangerous for agricultural ecosystems and causes huge economic losses. Phytoplasmas are insect-transmitted wall-less bacteria living in plants, only in the phloem tissues and in the emolymph of their insect vectors. They are able to manipulate several metabolic pathways of their hosts, very often without impairing their life. The molecular diversity described (49 'Candidatus Phytoplasma' species and about 300 ribosomal subgroups) is only in some cases related to their associated symptomatology. As for the other plant pathogens, it is necessary to verify their identity and recognize the symptoms associated with their presence to appropriately manage the diseases. However, the never-ending mechanism of patho-adaptation and the copresence of other pathogens makes this management difficult. Reducing the huge impact of phytoplasma-associated diseases in all the main crops and wild species is, however, relevant, in order to reduce their effects that are jeopardizing plant biodiversity.

European primary datasets of alien bacteria and viruses.

Bacteria and viruses are a natural component of Earth biodiversity and play an essential role in biochemical and geological cycles. They may also pose problems outside their native range, where they can negatively impact on natural resources, wildlife, and human health. To address these challenges and develop sustainable conservation strategies, a thorough understanding of their invasion related- factors is needed: origin, country and year of introduction, and pathways dynamics. Yet, alien bacteria and viruses are underrepresented in invasion ecology studies, which limits our ability to quantify their impacts and address future introductions. This study provides primary datasets of alien bacteria and viruses of plants and animals present in the European environment. The datasets contain expert-revised data on 446 taxa and their invasion related- factors across terrestrial and aquatic environments. Taxa information are complemented with spatial occurrences. The datasets provide a basis for collaborative initiatives to improve the collection of alien bacteria and viruses' data, and a starting point for data-driven conservation practices.

Containment of Phytoplasma-Associated Plant Diseases by Antibiotics and Other Antimicrobial Molecules.

Phytoplasmas are plant-pathogenic bacteria that infect many important crops and environmentally relevant plant species, causing serious economic and environmental losses worldwide. These bacteria, lacking a cell wall, are sensitive to antibiotics such as tetracyclines that affect protein synthesis mechanisms. Phytoplasma cultivation in axenic media has not been achieved for many strains; thus, the screening of antimicrobials must be performed using mainly in vivo materials. Some studies have investigated using in vitro phytoplasma-infected shoots, and several antimicrobials, including tetracyclines, have been tested. The screening of phytoplasma antimicrobials is important for the sustainable control of phytoplasma-associated diseases. The use of molecules with different modes of action such as ribosome inactivating proteins, plant hormones, and resistance inducers such as plasma-activated water, is advised, to avoid the use of antibiotics in agriculture and the possible emergence of resistant microbial strains.

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.