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Corn plants showing symptoms of midribs chlorosis, and leaf reddening, short internodes, ear proliferation, and plant growth reduction were collected in Peru from fields in nine localities in the provinces of Huancayo, Chupaca, and Jauja in the Junín region, and tested to verify phytoplasma presence and identity. Primers amplifying the phytoplasma ribosomal 16S and ribosomal protein genes were used. The phytoplasma presence was detected in symptomatic samples from all the surveyed areas. The sequencing of the obtained amplicons indicated the presence of ‘Candidatus Phytoplasma asteris’ and ‘Ca. P. pruni’-related strains. A BLASTn search of sequenced genes showed that the two ‘Candidatus Phytoplasma’ strains identified in corn shares 100% and 99.82% identity with the ‘Ca. P. asteris’ strains from maize and 99.92% and 99.55% with ‘Ca. P. pruni’-related strains, respectively. The RFLP analyses allowed to enclose these phytoplasma strains in the 16SrI-B and 16SrIII-J subgroups; however, the two phytoplasmas were, in some cases, present in mixed infection. The 16SrIII-J phytoplasma is for the first time reported associated with the maize bushy stunt disease and this represent a relevant information for the disease epidemiology towards its appropriate management in the affected area.

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.

“Huanglongbing” (HLB) is one of the most devastating diseases of citrus orchards worldwide. Samples from 183 citrus plants of different cultivars and rootstock/cultivar combinations, showing HLB symptoms in three Caribbean countries (Cuba, Jamaica, and Guadeloupe-France), were collected to verify the possible co-infection of ‘Candidatus Phytoplasma’ and ‘Candidatus Liberibacter’ species. The 64% of the samples resulted positive to the ‘Ca. L. asiaticus’ and the 27% to diverse ‘Ca. Phytoplasma’-related species, moreover about the 14% of the samples infected with ‘Ca. Liberibacter’ were also found positive to phytoplasmas, indicating the presence of mixed infection especially in the orchards located in Cuba. Moreover, in one of the samples from Jamaica mixed phytoplasma infection was detected. Moreover the detection of only phytoplasmas in 11 symptomatic citrus samples collected from Cuba and Guadeloupe without ‘Ca. Liberibacter’ detection, confirmed that the symptomatology cannot be the sole criterium to discriminate between the presence of the two pathogens, and molecular detection is necessary to identify single or mixed infections. Diaphorina citri insects collected from Cuba and Guadeloupe resulted infected with ‘Ca. L. asiaticus’ confirming its active role in the dissemination of the pathogen. Only one insect of the Cicadidae family, collected in Guadeloupe, was found positive for phytoplasma presence. Considering that the phytoplasmas belonging to some ‘Candidatus species’ were detected in the three countries in different citrus varieties, a relevant role as phytoplasma reservoir can be attribute to citrus orchards.

The possibility to grow on artificial media phytoplasmas isolated from micropropagated infected periwinkle shoots and from field-infected tissues is a recent achievement. Isolation and cultivation together with biochemical and biological characterization of some phytoplasmas was consistently obtained. Colonies of phytoplasmas belonging to diverse ribosomal groups were obtained such as those related to a ‘Candidatus Phytoplasma pruni’- related isolate from cassava plants with frog skin diseased, ‘Ca. P. asteris’ and ‘Ca. P. solani’ from diverse infected host plants such as grapevine and coconut palms. Antisera production and their preliminary diagnostic use was also achieved for some of these isolates. Further phytoplasmas were also isolated in liquid and solid media from date palm and citrus tissues. Seed transmission in corn of aster yellows phytoplasmas was confirmed by isolation from seedlings of viable phytoplasma cells.

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 % 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.

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The considerable economic losses in citrus associated with ‘Candidatus Liberibacter’ and ‘Candidatus Phytoplasma’ presence have alerted all producing regions of the world. In Chile, none of these bacteria have been reported in citrus species. During the years 2017 and 2019, 258 samples presenting symptoms similar to those associated with the presence of these bacteria were examined. No detection of ‘Ca. Liberibacter’ associated with “huanglongbing” disease was obtained in the tested samples; therefore, this quarantine pest is maintained as absent in Chile. However, 14 plants resulted positive for phytoplasmas enclosed in subgroups 16SrV-A (12 plants) and 16SrXIII-F (2 plants). Although they have been found in other plant species, this is the first report of these phytoplasmas in citrus worldwide.

The considerable economic losses in citrus associated with ‘Candidatus Liberibacter’ and ‘Candidatus Phytoplasma’ presence have alerted all producing regions of the world. In Chile, none of these bacteria have been reported in citrus species. During the years 2017 and 2019, 258 samples presenting symptoms similar to those associated with the presence of these bacteria were examined. No detection of ‘Ca. Liberibacter’ associated with “huanglongbing” disease was obtained in the tested samples; therefore, this quarantine pest is maintained as absent in Chile. However, 14 plants resulted positive for phytoplasmas enclosed in subgroups 16SrV-A (12 plants) and 16SrXIII-F (2 plants). Although they have been found in other plant species, this is the first report of these phytoplasmas in citrus worldwide.

In July 2017, a survey was conducted in a fig collection plot at Locorotondo (south of Italy) to investigate the possible presence of phytoplasmas in plants showing yellowing, deformed leaves, short internodes, mottling and mosaic. Samples were collected from symptomatic plants and tested by nested PCR assays using universal and specific primers to amplify the 16S rDNA of these prokaryotes. PCR results detected the presence of phytoplasma sequences in twenty plant samples that resulted clustering two phylogenetically distinct phytoplasmas, i.e., “Candidatus Phytoplasma asteris” and “Candidatus Phytoplasma solani” affiliated to 16SrI and 16SrXII ribosomal groups, respectively. The presence of phytoplasmas belonging to both ribosomal groups was confirmed with group specific quantitative PCR and RFLP assays on 16S ribosomal amplicons. Results of this study indicate for the first time the occurrence of phytoplasmas in fig; however, more work should be carried out to verify their association with the symptoms observed on diseased fig plants.

Lettuce is an important vegetable crop commercially produced in many countries and affected by several bacterial diseases. The presence of phytoplasmas in lettuce plants growing in open field in Calabria region (Southern Italy) and showing yellowing and leaf necrosis was detected by PCR assays with primers amplifying 16S rRNA gene sequence. The ‘Candidatus Phytoplasma asteris’ strain identified in the symptomatic samples was further amplified on amp, groEL, and leucine genes for its molecular characterization. This lettuce strain resulted scattered in the fields and was identified by RFLP analyses as 16SrI-B; a multigene characterization confirmed its ribosomal subgroup affiliation and the validity of these molecular markers for studying the geographic distribution of phytoplasma strains of this taxon.

The chrysanthemum is a popular ornamental flower all around the world for both cut flowers and potted plants. In recent days, bacterial infections are reducing in several cases its market value. Irregular flowering, yellowing and decline symptoms were observed at a commercial enterprise located in North Italy. To verify the presence of phytoplasmas, DNA was extracted from symptomatic and asymptomatic potted plants and subjected to specific polymerase chain reaction followed by restriction fragment length polymorphism analysis on phytoplasma 16S ribosomal gene. The tests were carried out three times in 7 months to verify the persistence of detected phytoplasmas over time. Phytoplasma classified in group 16SrV (elm yellows) were detected in all the examined samples independently from the presence of decline symptoms, while group 16SrXII (“stolbur”) phytoplasmas were only detected in the most severely declining plants. This latter phytoplasma strain was also characterized on tuf and leu genes by RFLP analyses and sequencing, respectively, to cross confirm its presence and identity. The detection of this phytoplasma in chrysanthemum plants is the first report in Italy, while it has already been identified in plants with malformed inflorescence in Europe, but not in other parts of the world.

In this study, two different cold atmospheric‐pressure plasmas are used for the production of plasma‐activated water (PAW). To evaluate the effectiveness of PAW as a possible means to control plant diseases, grapevines in an open field and a greenhouse are treated, evaluating qualitative and quantitative yield parameters, phytoplasma presence, and gene expression. The results show the capability of PAW to enhance plant defense mechanisms and, as demonstrated in the field trials, confirm its ability to improve the health status of the treated plants.

Nowadays, one of the main challenges is moving towards an eco-sustainable agriculture, able to preserve the food production through a reduced use of pesticides. Current global food sustenance by intensive agriculture is mainly based on economic crop monocultures and drastically reduces the biodiversity, increasing the yield losses due to the presence of biotic and abiotic stresses. A technology based on plasma activated water (PAW), characterized by the presence in liquid of reactive oxygen and nitrogen species, was tested to try to ensure yield stability also enhancing the plant resistance responses and to promote an eco-sustainable management of plant diseases. In PAW-treated micropropagated periwinkle shoots, periwinkle and grapevine plants, qRT-PCR and small RNAs high-throughput sequencing were used to analyse the differential expression of genes involved in the major plant defence pathways. The results indicate that PAW treatment enhances the plant defence responses and provide an encouraging framework for future applications in plant disease management programs.

Over 360 Hemiptera specimens were collected using sweep nets and hand-made aspirators from coconut palm fronds in six villages of Grand-Lahou. Eight families were identified including Aphrophoridae, Achilidae, Derbidae, Flatidae, Membracidae, Pentatomidae, Tropiduchidae, and Cicadellidae, the latter being the most abundant throughout the surveyed villages. PCR assays with primers targeting the 16S rRNA and the secA translocation protein genes yielded PCR amplicons from 216 out of 296 (73%) of the tested specimens of a newly identified cicadellid leafhopper, Nedotepa curta Dmitriev. PCR amplicons were purified, cloned and sequenced. The 16S rDNA and secA sequences from N. curta showed a 99% of sequence identity with those of the Cote d’Ivoire lethal yellowing (CILY) phytoplasma, member of subgroup 16SrXXII-B ‘Candidatus Phytoplasma palmicola’-related strain, previously identified in coconut-growing villages of Grand-Lahou. This suggested N. curta as a potential vector for the CILY phytoplasma. Four symptomatic coconut palms (7.4%) were found infected by a phytoplasma of group 16SrI, in mixed infection with the CILY phytoplasma (16SrXXII-B) in two palms, and alone in the other two palms, where the CILY phytoplasma was not detected. The 16SrI phytoplasma was also found in two N. curta specimens, and in the weeds Dalbergia saxatilis and Baphia nitida. Results indicating that mixed infection of both 16SrXXII-B and 16SrI phytoplasmas occurs in coconut palms affected by CILY in Grand-Lahou, and may impact disease management and control.

Different ornamental plants showing symptoms referable to phytoplasma presence and collected between 1993 and 2015 in various floricultural areas in north and south of Italy, enclosing Sicily, resulted to be infected by ‘Candidatus Phytoplasma asteris’-related strains, and after PCR/RFLP identification on 16Sr gene were assigned to 16SrI-B subgroup. These infected samples were employed for phytoplasma strain differentiation on tuf, groel, rp and amp genes. In particular, the 23 phytoplasma strains employed were from hydrangea (5), primula (3), gentian (2), petunia (2) and gerbera (1) samples showing flower virescence; from gladiolus samples both in vivo and in micropropagation (2) showing the “germs fins” symptomatology, from statice (2) with stunting and lack of flower production, from ranunculus (2) and carnation (4) with virescence and malformation of flowers. All the genes were amplified in nested PCR except amp gene. On tuf gene all the samples were amplified, and Tru1I RFLP analyses confirmed identical profiles with those of 16SrI group phytoplasmas, however on the other genes only samples from ranunculus, gladiolus in vivo, statice and hydrangea were amplified. On these genes the phytoplasmas were identical to each other and to reference strains belonging to 16SrI-B subgroups, and after RFLP analyses carried out with Tru1I and AluI they were further enclosed in the rpI-B and GroELI-III groups. Considering that these samples were collected in different Italian regions during 22 years, the relevant conservation in the studied genotypes can be perhaps linked to the presence of common leafhopper vectors, not always identified nor detected in the cultivation areas where the diseased plants were collected. It is important to underline that ‘Ca. P. asteris’ is the prevalent phytoplasma reported in flower cultivations worldwide, and its lack of genetic polymorphisms may also indicate a globalized trading of the pathogen together with his propagation material.

Liquidambar styraciflua was introduced in Bogotá (D. C.) Colombia, in the 1990s as an urban tree. Yellowing and deformation of the tree crowns began to appear in 2007, leading to the hypothesis of a possible phytoplasma infection. To detect and identify phytoplasmas, samples were collected from 21 liquidambar trees from Bogota in 2009, 2010 and 2011 and subjected to nested PCR, RFLP and sequencing analyses of 16S ribosomal DNA. Nested PCR assays with group-specific primers and RFLP analyses identified phytoplasmas belonging to the 16SrI-B (aster yellows), 16SrV-B (jujube witches' broom), 16SrVII-A (ash yellows), 16SrIX (pigeon pea witches' broom) and 16SrXII-A ("stolbur") groups/subgroups, in single or mixed infections. Sequence analysis and phylogenetic evaluation of samples with single phytoplasma infections confirmed the presence of phytoplasmas related to 'Candidatus Phytoplasma asteris', 'Ca. P. fraxini', and 'Ca. P. solani'. In 2010 and 2013 two further surveys involving 100 trees each time were carried out to estimate the prevalence of the disease: the average prevalence with severe symptoms was 28%, with mild symptoms, 56%, and with minor symptoms 16% of the trees. No symptomless trees were observed. Phytoplasma presence in liquidambar trees from Bogota carries economic, environmental and epidemiological consequences, and urgent measures should be implemented to avoid the spreading of the disease to other tree species and to the agricultural areas surrounding the city.

Surveys for the Cote d'Ivoire lethal yellowing (CILY) phytoplasma were conducted in eight severely CILY-affected villages of Grand-Lahou in 2015. Leaves, inflorescences and trunk borings were collected from coconut palms showing CILY symptoms and from symptomless trees. Total DNA was extracted from these samples and tested by nested polymerase chain reaction/RFLP and sequence analysis of the 16S rRNA, ribosomal protein (rp) and the translocation protein (secA) genes. The CILY phytoplasma was detected in 82.9% of the symptom-bearing palms collected from all the surveyed villages and from all the plant parts. Trunk borings were recommended as the most suitable plant tissue type for sampling. Results indicate that the CILY phytoplasma may have a westward spread to other coconut-growing areas of Grand-Lahou. CILY phytoplasma strains infecting coconut palms in the western region of Grand-Lahou exhibited unique single nucleotide polymorphisms on the rp sequence compared to the strains from the eastern region. Moreover, single nucleotide polymorphisms on the SecA sequence distinguished the CILY phytoplasma from the Cape St. Paul Wilt Disease phytoplasma in Ghana, and the Lethal Yellowing phytoplasma in Mozambique. (Resume d'auteur)

A phytoplasma-associated disease was iden-tified in Brassica campestris (sarson) plants during a survey conducted in Punjab province of Pakistan in 2014–2016. The symptomatic plants showed character-istic symptoms of phyllody and witches’ broom. Phytoplasma presence was detected by polymerase chain reaction on 16S ribosomal and tuf DNAs, follow-ed by RFLP analysis and sequence comparison of the 16S rRNA and tuf genes. The phytoplasma detected was classified in a new ribosomal subgroup designed 16SrIX-H. The phytoplasma presence in phloem tissues of symptomatic sarson samples was also confirmed through light microscopy and transmission studies to healthy plants through dodder and the leafhopper Orosius albicinctus. This is the first report of identifica-tion of 16SrIX-H subgroup phytoplasma associated with sarson and its natural vector in Pakistan.

Plasma activated water (PAW) possesses significant antimicrobial properties due to the synergistic effect of reactive oxygen and nitrogen species (RONS) produced by the plasma treatment. To test its efficacy as plant resistance inducer, trials were carried out in five Glera and one Chardonnay vineyards in grapevine plants preliminary tested for the presence and identity of phytoplasmas. The PAW treatments were performed for two years injecting into the plant vascular tissues 10-20 ml of PAW or sterile distilled water (SDW) as control in a total of 130 plants (105 treated with PAW and 25 with SDW). A relevant number of PAW treated symptomatic plants showed a reduction of the symptoms, an higher number of the bunches and a higher berry weight per plant, compared to the SDW treated. Moreover the molecular analyses after the two year-treatment show a decrease of phytoplasma infected plant number treated with PAW (35.5%) compared with the SDW treated ones (12,5 %).

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.

Cote 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.

The possibility to grow phytoplasmas in complex media as the mycoplasmas was ruled out for more than 40 years due to the inconsistency of the first isolation trials. The use of micropropagated infected periwinkle shoots first and tissues infected directly from the fields on artificial media was recently confirmed. Isolation and cultivation together with the first biochemical and biological characterization of some phytoplasmas were consistently obtained. Phytoplasmas were successfully grown from grapevine, cassava and coconut palm tissues severely infected and in some cases also from asymptomatic tissues from trees or plants growing in severely infected areas. Phytoplasmas from diverse ribosomal groups were isolated in particular from cassava with frog skin the detected prokaryotes in culture were molecularly identified as belonging to the 16SrIII group as in the original plants. Aster yellows and ‘stolbur’ phytoplasmas, group 16SrI and 16SrXII, respectively, were consistently grown from diverse host plants such as grapevine and coconut palms. Seed transmission in corn of aster yellows phytoplasmas was also confirmed by isolation from seedlings of viable cells. The growth and biochemical and biological characterization of these bacteria is therefore the most important recent step in the study of phytoplasmas that will allow to improve their knowledge and to carry out focused management in the field in order to reduce their impact on cultivated and wild crops worldwide.

In the last years, some reports of phytoplasmas presence in date palms have appeared indicating that these pathogens have a growing importance in this crop species. The present work was conducted to verify the possibility to isolate phytoplasmas infecting date palms in Saudi Arabia as alternative and/or complementary method to the amplification of 16Sr ribosomal gene. The comparison of phytoplasma detection from field infected materials and by isolation and colonies in culture indicates that the complexity of phytoplasma associated diseases is greater than believed and it is very likely that the symptomatology observed is the result of complex of microorganisms (mainly phytoplasmas) interaction. Further studies following this methodology would help in the better understanding of this complexity of phytoplasmas toward designing appropriate tools for the containment and management of these epidemic diseases to reduce their economic impact.

The development of methods for culturing phytoplasmas in artificial media suggested the evaluation of strategies to produce antibodies against a phytoplasma target. To this purpose, an aster yellows phytoplasma isolate obtained from field collected infected grapevine was used for the production of a polyclonal antiserum. It was then tested by ELISA and IFAS methods in order to check and evaluate its suitability for phytoplasma detection in plant samples.

Aster yellows phytoplasma strain Hyd35 (16SrI-B) in micropropagated periwinkle shoots in collection was used to produce infected plants in pots that were separated according to the diverse symptomatology i.e. phyllody and witches’ broom. Small RNA high-throughput sequencing (HTS) was then used to determine the small RNA pattern of these plants. Bioinformatics analysis revealed the presence of expression changes of different miRNA classes and the presence of phytoplasma derived small RNAs. These results could complement previous studies and serve as a starting point for small RNA omics in phytoplasma research.

Surveys in and nearby vineyards in some Italian provinces were carried out to detect alternative host plants and possible insect vectors of phytoplasmas detected in grapevines. Aster yellows is the prevalent phytoplasma detected in both beside the 16SrXII-A and 16SrV-C phytoplasmas. Moreover other phytoplasmas belonging to diverse ribosomal groups were identified indicating a relevant epidemiological role of spontaneous vegetation and possible new insect vectors in the grapevine yellows.

The focus of this research was the development and evaluation of different complex liquid and solid media for the isolation and growth of phytoplasma strains infecting grapevine plants. Previously reported media supporting phytoplasma isolation are commercial and not easy to modify in order to improve performance and selectivity towards obtaining pure cultures of 'Candidatus Phytoplasma' species. Three media (Piv®, CB and MB) were therefore evaluated for phytoplasma isolation and colony formation under microaerophilic growing conditions, using grapevine canes from plants showing yellows symptoms, and infected by "flavescence dorée", "bois noir" and aster yellows phytoplasmas as sources. The newly developed methodology was applied for two years at three sample collection times. Broad applicability and a good repeatability in supporting phytoplasma colony formation were obtained in Pivs® and CBs media. While the MB medium did not support phytoplasma isolation and growth, the CB media support a phytoplasma growth comparable to the one obtained in the previously reported media. This medium has the advantage of a formulation that allow its modification to implement specificity towards selective phytoplasma growth. Moreover preliminary trials on serial dilutions and tetracycline addition confirmed some phytoplasma growth behaviours.