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Two of the most common leafhoppers present in Chile are the Cicadellidae Paratanus exitiosus and Bergallia valdiviana. They commonly occur in vineyards of central Chile, including some vineyards infected by phytoplasmas. The present study demonstrates that P. exitiosus and B. valdiviana can transmit 16SrIII-J phytoplasmas to grapevine and periwinkle plants. This provides improved understanding of the 16SrIIIJ phytoplasma epidemiology in Chilean vineyards.

La trasmissione dei fitoplasmi attraverso il seme è una questione dibattuta e controversa a causa della ridotta conoscenza sulle connessioni tra pianta-madre ed embrione a livello floematico. I fitoplasmi sono stati rinvenuti in diverse parti della pianta compresi gli organi riproduttivi, l’embrione ed i frutti e la produzione di semi in piante infette risulta ridotta. Recenti studi hanno evidenziato che le infezioni tardive, in particolare nelle colture erbacee, possono consentire una normale produzione di semi. Inoltre, anche in presenza di anomalie in fiori e frutti, i semi prodotti possono essere vitali e con buona capacità di germinazione. Nella sperimentazione si sono utilizzati semi di Sesamum indicum, Brassica napus, Solanum lycopersicum e Zea mays prodotti da piante-madri infette da fitoplasmi. Le piantine ottenute sono state saggiate a 40 e a 90 giorni dalla germinazione ed il 90% dei campioni di S. indicum è risultato positivo per fitoplasmi dei gruppi ribosomici 16SrI, 16SrII e 16SrXII-A sia a 40 che a 90 giorni. L'85% dei campioni di B. napus è risultato positivo per i fitoplasmi appartenenti a gruppi ribosomici 16SrI e 16SrXII-A a 40 e 90 giorni. Il 20% dei campioni di S. lycopersicum è risultato positivo per fitoplasmi dei gruppi ribosomici 16SrI e 16SrXII-A a 40 giorni dalla germinazione mentre solo il 10% è risultato positivo a 90 giorni dalla germinazione. Da queste piante di pomodoro sono state ottenute bacche i cui semi hanno dato origine a piante di seconda generazione risultate a loro volta positive (12%) a fitoplasmi appartenenti ai gruppi ribosomici 16SrI e 16SrXII-A, indicando la presenza di fitoplasmi anche nella seconda generazione di piante. Il 20% dei campioni di Z. mays è risultato positivo a fitoplasmi 16SrI e 16SrXII-A a 40 giorni dalla germinazione e il 10% è risultato ancora positivo a 90 giorni. Da alcuni di questi campioni di mais è stato eseguito l'isolamento dalle nervature, in substrato CB: da alcune piante (mais 3E, 4.5, 4E, 1.1, 1.2 e 1D), positive a 90 giorni, è stato possibile ottenere colonie di diverse dimensioni e forma, alcune delle quali sono risultate positive alla presenza di fitoplasmi 16SrI (“aster yellows”). Dai campioni di mais 3E, 4.5 e 4E si sono ottenute colonie anche da brodi mantenuti per 7 mesi a 25°C dopo l'isolamento iniziale. Dal campione di mais 3E si sono ottenute alcune colonie di piccole dimensioni poi sottoposte a passaggi di purificazione da substrato liquido a solido; anche queste colonie sono risulte positive ad “aster yellows”. Si sono ottenute colonie da almeno 3 passaggi successivi da substrato liquido a solido eseguiti ogni 5 giorni. I risultati indicano la vitalità del fitoplasma “aster yellows”, isolato da piantine di mais ottenute da seme infetto e mantenute in ambiente privo di insetti e confermano la trasmissione di fitoplasmi vitali attraverso il seme.

Introduction Decline and yellows are diseases reported since longtime in several European hazelnut (Corylus avellana L.) growing areas and were associated since 20 years to 16SrX group phytoplasma presence (1). More recently in stunted, but also in asymptomatic plants a 16SrIII-B phytoplasma was identified in Oregon (USA) (2). Hazelnut infection by phytoplasmas appears therefore a puzzling issue. During a survey in the Campo Experimental Maquehue, Universidad de La Frontera, Temuco, hazelnut plants cv. Barcelona 12 year-old showing leaf and catkin malformation, and yellowing in some branches were sampled to verify phytoplasma presence. Materials and Methods Leaf and small branch samples from symptomatic and asymptomatic hazelnut plants were collected at the beginning of the Spring (December) 2016 and, after a chloroform-based DNA extraction of phloematic tissues, they were subjected to nested PCR on the 16S ribosomal gene (3) followed by RFLP analyses and direct amplicon sequencing for phytoplasma identification and phylogenetic clustering, respectively. Results and Dıscussıon Only the symptomatic samples resulted positive to phytoplasma presence and RFLP analyses on 500 bp amplicons (M1/M2 primers) indicate the presence of 16SrI, 16SrIII, and 16SrV phytoplasmas. In particular, in samples showing yellow leaves 16SrI and 16SV phytoplasmas were identified in single or mixed infection; moreover RFLP analyses on amplicons obtained with the 16SrI group specific primers show the presence in the 16SrI phytoplasmas of a Tru1I profile that was different from all those reported so far. In the small branches with yellow in the leaflets surrounding the young nuts 16SrIII phytoplasmas were preliminary identified on 500 bp amplicons. The sequencing confirmed the phytoplasma clustering and the polymorphism determined by RFLP analyses. The yellowing observed in Chilean hazelnut was believed to be due to the presence of nutritional disorders related to the quite new environment for the crop. The finding of phytoplasmas is prompting to verify now their relationship with the symptomatology observed. The detection of several phytoplasmas in Chilean hazelnuts confirms the species susceptibility to these bacteria (1, 2) and their possible influence on the plant metabolism. Vegetative propagation together with still unknown insect vector(s) could be the main responsible for presence of several diverse phytoplasmas in the relatively restricted area where the hazelnut is starting to be grown in Chile.

In several growing areas of Northern Italy cherry is affected by a severe decline that leads plants to die in two-weeks/one month maximum. From the beginning of the summer the affected plants show leaves of smaller size, with chlorosis, reddening, curling aspect and premature fall; young branches also show some lack of lignifications. Preliminary tests excluded presence of several biotic or abiotic agents; to verify the possible phytoplasma association with the disease nucleic acid extraction was performed from leaf midribs as well as from phloem scrapes of trunks from declining trees. Molecular analyses carried out on samples from declining cherry plants collected in different orchards from 2002 to 2004 allowed identification of phytoplasmas belonging to different ribosomal subgroups such as 16SrV-B; 16SrIII-B and 16SrXII-A (stolbur). Since the area monitored was limited and phytoplasma detection was achieved in both hilly and sandy soil locations on plants usually older than 5–6 years, it appears possible that infection was related to the health status of propagation material employed for plantations.

The frequency of normoblastemia in dogs receiving chemotherapy is unknown. To provide this information, we calculated the percentage and number of nucleated erythrocytes (nRBCs) in blood of dogs treated for lymphoma (n = 284), mast cell tumour (n = 40) or carcinoma (n = 46). Relative normoblastemia (1 or5%) and absolute normoblastemia (0.1 or0.4 × 10

IN THE LAST DECADE THE ECONOMIC IMPORTANCE OF HYDRANGEA DISEASES ASSOCIATED WITH VIRUS AND PHYTOPLASMAS IN ITALY INCREASED THEREFORE EPIDEMIOLOGICAL SURVEYS WERE CARRIED OUT IN LIGURIA AND LAZIO REGIONS IN 2010 AND 2011. TO DETECT VIRUS INFECTIONS MECHANICAL INOCULATIONS ON HERBACEOUS PLANTS, ELECTRON MICROSCOPE OBSERVATIONS OF “LEAF-DIP” PREPARATIONS, SEROLOGY (PAS-ELISA AND ISEM TECHNIQUES) WERE EMPLOYED; TO VERIFY PHYTOPLASMAS PRESENCE AND TO DETERMINE THEIR IDENTITY, SAMPLES WERE TESTED BY NESTED-PCR, FOLLOWED BY RFLP ANALYSES ON 16S RIBOSOMAL GENE. IN 2011, HYDRANGEA PLANTS SHOWINGS STUNTING, FLOWER VIRESCENCE AND PHYLLODY, YELLOWING, NECROSIS AND REDNESS OF THE LEAF EDGE, WERE COLLECTED IN TWO COMMERCIAL GREENHOUSES OF “PIANA DI ALBENGA” (LIGURIA REGION). ALL SAMPLES (BELONGING TO THREE HYBRIDS) WERE INFECTED BY HYDRANGEA RINGSPOT VIRUS (HRSV). ONE OF THESE HYDRANGEA PLANTS, SHOWING ALSO FLOWER VIRESCENCE AND RED EDGES OF LEAVES, WAS INFECTED BY PHYTOPLASMAS BELONGING TO GROUP 16SRI-B (‘CANDIDATUS PHYTOPLASMA ASTERIS’). FURTHER RFLP CHARACTERIZATION OF THIS PHYTOPLASMA STRAIN ON THE GROEL GENE WITH TRU1I AND ALUI ALLOW ASSIGNING IT TO GROELI SUBGROUP III, REPORTED SO FAR ON SEVERAL EUROPEAN COUNTRIES. DURING INSPECTIONS PERFORMED IN THE BIENNIUM 2010-2011, SEVERE VIRUS-LIKE SYMPTOMS WERE OBSERVED IN ALMOST ALL HYDRANGEA PLANTS GROWING IN BOLSENA CITY (LAZIO REGION) SUCH AS STUNTING, LEAF AND FLOWER MALFORMATIONS, MOSAIC, CHLOROTIC AND NECROTIC MOTTLE, COLOUR-BREAKING ON PETALS. VIROLOGICAL TESTS REVEALED THE PRESENCE OF CUCUMBER MOSAIC VIRUS (CMV) IN THREE PLANTS CHARACTERIZED BY STUNTING, LEAF MOSAIC IN MALFORMED LEAVES AND FLOWER COLOUR-BREAKING. THE PRESENCE OF ELM MOTTLE VIRUS (EMOV; SYN. HYDRANGEA MOSAIC VIRUS) IN TWO PLANTS WITH SYMPTOMATIC LEAVES WAS ALSO DETECTED. MECHANICAL TRANSMISSION OF VIRUSES FROM HYDRANGEA WAS OBTAINED IN GOMPHRENA GLOBOSA WITH ALL THE IDENTIFIED VIRUSES BUT EMOV; THIS LATTER INDUCED SYSTEMIC INFECTION IN CHENOPODIUM QUINOA, HOWEVER ONLY SEROLOGICAL ANALYSES ALLOW TO IDENTIFY THE DIVERSE VIRUSES. IN 2011 ONE PLANT SHOWING GROWTH REDUCTION, FLOWER VIRESCENCE AND PHYLLODY, AND WITH ASYMPTOMATIC LEAVES WAS FOUND; MOLECULAR ANALYSES ALLOW TO IDENTIFY THE PRESENCE OF PHYTOPLASMAS BELONGING TO RIBOSOMAL SUBGROUP 16SRXII-A “STOLBUR”. FURTHER STRAIN CHARACTERIZATION CARRIED OUT ON STAMP AND TUF GENES CONFIRMED THE PRESENCE OF “STOLBUR” PHYTOPLASMAS. RFLP ANALYSES WITH TRU1I ON STAMP GENE SHOW THAT THE STRAIN INFECTING HYDRANGEA BELONGS TO ONE OF THE TWO GROUPS DIFFERENTIABLE IN “STOLBUR” PHYTOPLASMAS IN SOUTHERN EUROPE. LEAFHOPPERS PRESENT ON THIS LATTER PHYTOPLASMA-INFECTED PLANT WERE CAUGHT AND IDENTIFIED AS LAODELPHAX STRIATELLUS, ANACERATOGALLIA SP., EMPOASCA DECIPIENS, EMPOASCA SP., ASYMMETRASCA DECEDENS, ZYGINIDIA PULLULA. DNA FROM IDENTIFIED SPECIMENS MAINTAINED IN 100% ETHANOL WAS EXTRACTED AND TESTED BY NESTED-PCR/RFLP ANALYSES ON 16S RIBOSOMAL GENE AND TUF GENES. ‘CA. P. ASTERIS’-RELATED PHYTOPLASMAS WERE IDENTIFIED IN L. STRIATELLUS WHILE “STOLBUR” (16SRXII-A) PHYTOPLASMAS WERE PRESENT IN ANACERATOGALLIA SPP. CMV AND EMOV HAVE BEEN DETECTED IN THIS SPECIES FOR THE FIRST TIME IN ITALY; IN ADDITION, UNTIL NOW, ONLY PHYTOPLASMAS BELONGING TO SUBGROUP 16SRI-B (ASTER YELLOWS) HAVE BEEN FOUND INFECTING HYDRANGEA IN ITALY AND WORLDWIDE. HOWEVER 16SRXII-A PHYTOPLASMAS WERE ONLY REPORTED IN HYDRANGEA IN BULGARIA MORE THAN 15 YEARS AGO IN MIXED INFECTION WITH ASTER YELLOWS, THIS IS THEREFORE THE FIRST CONFIRMATION OF STOLBUR PHYTOPLASMA PRESENCE ASSOCIATED WITH VIRESCENCE OF H. MACROPHYLLA. EPIDEMIOLOGICAL SURVEYS WERE CARRIED OUT IN LIGURIA AND LAZIO REGIONS IN 2010 AND 2011 TO DETECT VIRUS AND PHYTOPLASMA INFECTIONS IN HYDRANGEA MACHROPHYLLA BY APPLYING (FOR VIRUS PRESENCE) MECHANICAL INOCULATIONS ON HERBACEOUS PLANTS, ELECTRON MICROSCOPE OBSERVATIONS OF “LEAF-DIP” PREPARATIONS, SEROLOGY (PAS-ELISA AND ISEM TECHNIQUES) AND (FOR PHYTOPLASMA PRESENCE) NESTED-PCR, FOLLOWED BY RFLP ANALYSES ON 16S RIBOSOMAL GENE. HYDRANGEA PLANTS SHOWINGS STUNTING, FLOWER VIRESCENCE AND PHYLLODY, YELLOWING, NECROSIS AND REDNESS OF THE LEAF EDGE, COLLECTED IN TWO COMMERCIAL GREENHOUSES OF “PIANA DI ALBENGA” (LIGURIA REGION), WERE INFECTED BY HYDRANGEA RINGSPOT VIRUS (HRSV); ONE OF THESE HYDRANGEA PLANTS, SHOWING ALSO FLOWER VIRESCENCE AND RED EDGES OF LEAVES, WAS INFECTED BY PHYTOPLASMAS BELONGING TO GROUP 16SRI-B (‘CANDIDATUS PHYTOPLASMA ASTERIS’) GROELI SUBGROUP III. ALMOST ALL HYDRANGEA PLANTS GROWING IN BOLSENA CITY (LAZIO REGION) SHOWED SEVERE SYMPTOMS: CUCUMBER MOSAIC VIRUS (CMV) INFECTED THREE PLANTS CHARACTERIZED BY STUNTING, LEAF MOSAIC IN MALFORMED LEAVES AND FLOWER COLOUR-BREAKING; ELM MOTTLE VIRUS (EMOV; SYN. HYDRANGEA MOSAIC VIRUS) INFECTED TWO PLANTS WITH SYMPTOMATIC LEAVES. ONE PLANT SHOWING GROWTH REDUCTION, FLOWER VIRESCENCE AND PHYLLODY WAS FOUND INFECTED BY PHYTOPLASMAS BELONGING TO RIBOSOMAL SUBGROUP 16SRXII-A “STOLBUR”. LEAFHOPPERS PRESENT ON THIS LATTER PHYTOPLASMA-INFECTED PLANT WERE CAUGHT AND IDENTIFIED AS LAODELPHAX STRIATELLUS, ANACERATOGALLIA SP., EMPOASCA DECIPIENS, EMPOASCA SP., ASYMMETRASCA DECEDENS, ZYGINIDIA PULLULA. ‘CA. P. ASTERIS’-RELATED PHYTOPLASMAS WERE IDENTIFIED IN L. STRIATELLUS WHILE “STOLBUR” (16SRXII-A) PHYTOPLASMAS WERE PRESENT IN ANACERATOGALLIA SPP. CMV AND EMOV HAVE BEEN DETECTED IN THIS SPECIES FOR THE FIRST TIME IN ITALY; IN ADDITION, UNTIL NOW, ONLY PHYTOPLASMAS BELONGING TO SUBGROUP 16SRI-B (ASTER YELLOWS) HAVE BEEN FOUND INFECTING HYDRANGEA IN ITALY AND WORLDWIDE. HOWEVER 16SRXII-A PHYTOPLASMAS WERE ONLY REPORTED IN HYDRANGEA IN BULGARIA MORE THAN 15 YEARS AGO IN MIXED INFECTION WITH ASTER YELLOWS, THIS IS THEREFORE THE FIRST CONFIRMATION OF STOLBUR PHYTOPLASMA PRESENCE ASSOCIATED WITH VIRESCENCE OF H. MACROPHYLLA.

Background: Information about the possible role of serum activity of creatine kinase (CK) isoenzymes as a biomarker for septicemia and asphyxia in newborn foals is not available. Objectives: To evaluate the activity of CK isoenzymes in healthy newborns and in septicemic or asphyctic foals. Methods: Electrophoretic separation of CK isoenzymes (CK-MM, CK-MB, CK-BB) and macroenzymes (Macro-CKI and Macro-CK2) was performed on sera from healthy foals sampled 30 minutes after birth, at 3, 12, 24 hours, and daily until day 7. Sera from 9 septicemic and 5 asphyctic foals were also examined at admission and during the follow up. The results were compared with those of age-matched controls. For both healthy and sick foals, differences among the sequential time samplings were also assessed. Results: In healthy foals, total CK activity significantly increased in the first day then decreased on day 2. CK-BB is the main isoenzyme at birth and up to day 7, except at 3 and 12 hours, when CK-MM is the prevalent isoenzyme. CK-MB and macroenzymes are negligible. Compared with age-matched controls, asphyctic foals have increased CK-MM activity, whereas electrophoretic changes in septicemic foals are variable and generally mild. During the follow up, isoenzyme activities normalized in both groups of sick foals. Conclusions: The proportions and activities of the different isoenzymes vary with age, with CK-BB being the main serum isoenzyme in most samplings. Thus it is important to compare the results of sick foals with those of age-matched controls. This comparison reveals severe CK-MM increases in asphyctic foals and variable changes in septicemic foals.

Identification of phytoplasmas associated with carrot yellows in Serbia allow to identify 16SrI-A and 16SrI-B subgroups (Duduk et al., Annals of Applied Biology, 154, 219-229. 2009). However, PCR amplification of 16SrDNA followed by RFLP analysis, cloning and sequencing showed clearly presence of interoperon heterogeneity in one of the samples: two different sequences were obtained associated with two different RFLP profiles. Homology comparison among the sequences clustered in 16SrI-B clade showed that cloned sequences of the strain are closer to some other 16SrI-B strain sequences than to each other. Moreover phylogenetic analyses of the two operons showed that, while one operon is clustering in the 16SrI-B clade, the other operon is clustering out of it. The two operons of the same phytoplasma can be affiliated to different 16SrI subgroups according to RFLP analyses and this is supported also by phylogenetic analyses. Therefore, additional genes such as the l22 and s3 ribosomal protein genes, the tuf gene coding the elongation factor Ef-Tu, the putative aa kinase gene and ribosomal recycling factor gene, and a phytoplasma DNA helicase gene were studied to molecularly characterize this aster yellows strain from carrot. The RFLP and sequence analyses of PCR amplified ribosomal protein genes clearly showed that the strain is different from those affiliated with rpI-B and from all other strains in rpI subgroup tested. This strain was also differentiable from all other strains by RFLP analyses of putative aa kinase gene and ribosomal recycling factor gene, while analyses of tuf gene and of DNA helicase gene did not supported the difference and did not show any polymorphism, respectively. The presence of 16S rRNA interoperon sequence heterogeneity is not uncommon in phytoplasmas, and although the difference in homology between two operons is relatively small, when differences occur in restriction sites, misidentification or assignment of the same phytoplasma to two different 16S rRNA subgroups is possible. However, the use of other genes present as single copy in the phytoplasma genome can be helpful in discriminating when different phytoplasma populations are present in mixed infection from the presence of interoperon sequence heterogeneity.

The production losses associated with European stone fruit yellows (ESFY, 16SrX-B) phytoplasmas in Italian plum orchards reach up to 40% in Japanese plum. During six years a trial was carried out in Vignola area (Northern Italy) to assess the susceptibility of several plum varieties to the infection by ESFY phytoplasmas. In the surrounding of the experimental orchard ESFY presence was identified in declining cherry; Cacopsylla pruni was negative to phytoplasma presence, while Fieberiella florii specimens resulted infected by 16SrX-B phytoplasmas (Landi et al., Bulletin of Insectology, 60, 163-164. 2007), providing evidence for the pathogen presence in the environment. Varieties, cultivars and new selections of European and Japanese plum employed were grafted on Myrabolan 29C, and derived from commercial nurseries and from breeding programs. Plants were evaluated in 2-4 plots of four plants each. Yearly monitoring by visual inspection and PCR/RFLP identification of phytoplasmas allowed verifying the ESFY phytoplasma presence in the orchard since the first year of plantation. After a scattered phytoplasma presence detected in the year of plantation mainly in asymptomatic plants, an increasing ESFY presence in both symptomatic and asymptomatic plums was observed in subsequent years. After the six years monitoring among the 30 Japanese plums eight selections showed ESFY symptoms or pathogen presence in the 50% of plants, and nine selections showed 20% of infection. Only nine among cultivars and selections -Bragialla, Brarossa, Fortune, Ruby Crunch, n. 89.030.020, n. 89.030.031, n. 89.036.131, n. IFF/260, and n. IFF271- showed absence of both symptoms and pathogen. Although the majority of the 35 European types of plum was not symptomatic, some of the genotypes -Rheingold, Valcean, Valerie, n. 3018– showed one to three symptomatic plants ESFY-infected each, and one asymptomatic selection, n. 1474, was also positive to the sane phytoplasma in one plant.

To study 16Sr I-C phytoplasma (clover phyllody subgroup) transmission in cultivated areas where symptomatic strawberry plants show autumnal flowers with sepals instead of petals (virescence), and produce fruits with leaf-like tissues instead of seeds (phyllody), samples from 71 plants and from 2811 individuals of Trialeurodes vaporariorum (Westw.) and Empoasca spp. insects were analysed, using molecular tools. Four symptomatic strawberry plantlets collected in the autumn of 2002, and one Trialeurodes vaporariorum (Westw.) sample collected in June 2003, tested positive for 16SrI-C subgroup phytoplasmas. The plant and insect samples collected after autumn 2003 in the same two fields where the new strawberry plants showed absence of virescence and phyllody symptoms, tested negative for 16Sr I-C phytoplasmas using molecular analyses.

Prunus species are prone to infections by viruses and phytoplasmas, against which no effective cure exists for already infected plants in the field. Phytoplasmas infecting fruit trees can cause severe symptoms and are considered as quarantine organisms in Europe and North America. However, detection often is hampered by their irregular distribution in host plants. In the frame of phytosanitary measures a sensitive, specific and quick detection system would be highly desirable for routine detection, mainly to avoid the use of infected planting material. The development of improved strategies for the production of elite plants of phytoplasma-free stone fruit cultivars to be used for the production of certified elite propagation material was achieved. In vitro thermotherapy and meristem culture to eliminate pathogens from stone fruit plants were applied and protocols were validated for survival rates of shoots and plants and on their effectiveness for pathogen elimination. To gain time and to confirm the elimination of these pathogens from planting material by the applied in vitro treatments, improved detection methods were tested on micropropagated material soon after the treatment. The use of diverse general primers in bi-nested PCR allows to detect phytoplasmas belonging to different groups. Broad spectrum PCR are advisable for fruit tree material in micropropagation when the sanitary status of the mother plants is not known, while specific PCR primers could be employed to detect the presence of a known phytoplasma.

Objective To assess whether alpha‐1‐acid glycoprotein (AGP) can be detected on the membrane of feline circulating leucocytes. Design The presence of AGP on circulating leucocytes was investigated in both clinically healthy cats and cats with different diseases. A group of feline coronavirus (FCoV)‐positive cats, comprising cats with feline infectious peritonitis (FIP) and cats not affected by FIP but seropositive for FCoV, were included in this study because the serum concentration of AGP increases during FCoV infection. Procedure Flow cytometry (using an anti‐feline AGP antibody), serum protein electrophoresis, routine haematology and measurement of the serum AGP concentration were performed using blood samples from 32 healthy cats (19 FCoV‐seropositive), 13 cats with FIP and 12 with other diseases (6 FCoV‐seropositive). The proportion of cats with AGP‐positive leucocytes in the different groups (e.g. controls vs sick; FIP vs other diseases, etc.) or in cats with different intensities of inflammatory response was compared using a Chi‐square test. Results AGP‐positive leucocytes were found in 23% of cats. Compared with controls, the proportion of patients with positive granulocytes and monocytes was higher among sick cats (especially cats with diseases other than FIP) and cats with high serum AGP concentration, but not in cats with leucocytosis or that were FCoV‐seropositive. Conclusion AGP‐positive leucocytes can be found in feline blood, especially during inflammation. Conversely, no association between AGP‐positive leucocytes and FIP was found. Further studies are needed to elucidate the mechanism responsible for this finding and its diagnostic role in cats with inflammation.

A survey to verify phytoplasma presence was carried out in North-Central Italy on samples from peach and cherry orchards using the same PCR/RFLP methodology for both species. Direct and nested PCR with primers amplifying phytoplasma ribosomal or ribosomal plus spacer DNA were applied. Primers specific for different phytoplasma groups already detected in fruit trees, as well as primers amplifying a short (500 bp) ribosomal phytoplasma DNA fragment, were used to increase detection sensitivity. RFLP analysis for phytoplasma identification was then performed. Three nectarine cultivars showing some trees with delay in flowering and in fruit production were tested together with asymptomatic plants in April/May 1999. Only nested PCR with phytoplasma group specific or M1/M2 primers provided positive results. RFLP analyses identified 16SrX-A, 16SrI, and 16SrXII-A phytoplasmas. In one plant a mixed infection of 16SrI and 16SrX-A phytoplasmas was detected. No clear relationship was found among symptoms, peach variety and phytoplasma presence. The sweet cherry variety Prime Giant showed a decline shortly after plantation; in samples tested during spring and summer phytoplasmas of 16SrI-B, 16SrX-B; 16SrX-C, 16SrXII-A and 16SrIII groups were often detected therefore a relationship between cherry decline and phytoplasmas could be possible.

Homocysteine (Hcy) is a sulphur-containing intermediate product of methionine metabolism. Hcy is further metabolised by vitamin B12- or B6-dependent pathways (Stanger and others 2003). In people, hyperhomocysteinaemia is an independent risk factor for cardiovascular, thrombotic, neurodegenerative, pregnancy-associated diseases (Stanger and others 2003, Refsum and others 2004), and it is a sensitive marker of vitamin B12 deficiency consequent to malabsorption (Papa and others 2001), although its specificity is low, since Hcy increases in several pathophysiologic conditions, including folate deficiency (Stanger and others 2003). No information about the association between hypocobalaminaemia and hyperhomocysteinaemia in dogs is available. The aim of this study was to assess whether serum levels of Hcy are increased in dogs with gastrointestinal disorders and hypocobalaminaemia compared with dogs with gastrointestinal disorders not associated with hypocobalaminaemia. Toward this aim, 17 serum samples from dogs with symptoms consistent with gastrointestinal disease, referred to the author's institutions for diagnostic or therapeutic procedures associated with gastrointestinal disease were analysed. According to the guidelines of the Animal Care and Use Committee of our institution, it was not necessary to require a formal approval for the study, since all the dogs were sampled under informed consent of the owner, during diagnostic procedures. The only inclusion criterion was the presence of clinical (vomiting, diarrhoea, weight loss), and histopathological findings (presence of inflammatory infiltrates of different type and magnitude in the intestinal wall) consistent with gastrointestinal disorders potentially associated with malabsorbtion and hypocobalaminaemia. Based on a previous report …

Additional index words. nested PCR, RFLP analyses, disease, Zizyphus jujuba Abstract. Jujube (Zizyphus jujuba Mill.) witches' broom (JWB) is the most important disease in the areas of jujube cultivation in China, where it occurs every year. Micropropa- gated shoots of the three most important cultivars ('Lizao', 'Junzao', and 'Muzao') in the National Jujube Gene Pool, collected at the Pomology Institute of Shanxi province, were tested for the presence of phytoplasmas. Phytoplasma ribosomal (16Sr) general and specific primer pairs were used in direct or nested polymerase chain reaction (PCR). Positive results were obtained only from symptomatic micropropagated samples of 'Lizao' and from phytoplasma controls. Restriction fragment length polymorphism (RFLP) analyses of PCR products with several restriction enzymes revealed that the phytoplasmas infecting the symptomatic plants belong to the 16SrRNA group V subgroup B. The positive correlation between symptoms and the presence of phytoplasmas was verified in tissue culture. Samples from apparently healthy shoots of 'Junzao', 'Muzao', and 'Lizao' were free of phytoplasmas. In 1997, symptoms of witches' broom were detected in some 'Lizao' jujube plants in an experimental field at the National Jujube Gene Pool (NJGP) at the Pomology Institute of Shanxi Province (PISP), China. Since the dis- ease was associated with the presence of phytoplasma (Zhu et al., 1998), micropropa- gated 6-month-old shoots of the commercially important cultivars Lizao, Junzao, and Muzao derived from this field were used for DNA extraction to verify the presence of these prokaryotes.

Apple, pear and apricot trees showing phytoplasmaassociated symptoms from Ankara and Isparta provinces were sampled andinvestigated to verify the presence of phytoplasma-associated diseases. Totally31 samples were tested with phytoplasma universal and group specific primersand the PCR products were restricted using Tru1I,RsaI and SspI endonucleases. Different RFLP profiles were obtained andselected samples were directly sequenced. The apple samples were found infectedwith 16SrX-A (‘Candidatus Phytoplasmamali’), while the majority of the pear samples were infected with 16SrX-A and16SrX-C subgroup phytoplasmas in mixed infection. The 16SrX-B (‘Candidatus Phytoplasma prunorum’),16SrX-C (‘Candidatus Phytoplasmapyri’) and mixed infection of 16SrX-A/16SrX-C and 16SrX-C/16SrI (aster yellows)were detected in the apricot samples. In this study the 16SrI phytoplasmas inapricot and the mixed phytoplasma infections in pear and apricot trees were detectedin Turkey for the first time., Ankara ve Isparta illerinden fitoplazma enfeksiyonubenzeri belirti gösteren elma, armut ve kayısı ağaçlarından fitoplazma varlığınınsaptanmasına yönelik örnekleme yapılmıştır. Toplamda 31 örnek universal ve grupspesifik fitoplazma primerleri ile PCR’a tabi tutulmuş ve elde edilen PCRürünleri Tru1I, RsaI and SspIendonükleazları ile kesilmiştir. Elma örnekleri 16SrX-A (‘Candidatus Phytoplasma mali’) ile enfekteli bulunurken armutörneklerinin çoğunluğunda 16SrX-A ve 16SrX-C alt gruplarıyla karışık enfeksiyonsaptanmıştır. Kayısı örneklerinde 16SrX-B (‘CandidatusPhytoplasma prunorum’), 16SrX-C (‘CandidatusPhytoplasma pyri’) ve 16SrX-A/16SrX-C ve 16SrX-C/16SrI (aster yellows) alt gruplarınınkarışık enfeksiyonu saptanmıştır. Bu çalışma ile kayısıda 16SrI, armutta vekayısıda karışık fitoplazma enfeksiyonları Türkiye’de ilk kez saptanmıştır.

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.

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.

Coconut lethal yellowing (LY) is the single most important disease presently affecting the coconut production worldwide. Symptomatic and asymptomatic coconut plants were sampled in some selected areas to verify the identity of phytoplasmas associated with LY in Cuba. Diverse phytoplasma ribosomal groups were detected in the samples from symptomatic palms such as 16SrXII, 16SrVII, and 16SrI. In several other palms 16SrIV-A subgroup phytoplasmas were identified, in the groEL gene the only positive plant from Pilón resulted diverse from the others and identical to the 16SrIV-A strains detected in Jamaica LY infected coconut palms. This is the first record of occurrence of 16SrI, -VII and -XII groups in coconut palms in Cuba.

The transmission of phytoplasmas by seed was reported in several herbaceous and some woody plant species. The seedlings were usually analyzed for the presence of phytoplasmas in different stages of growth by nested-PCR/RFLP analysis and phytoplasmas belonging to different ribosomal groups according with species and geographical distribution were detected. The phytoplasma isolation from corn seedlings confirms the seed transmission of viable phytoplasma cells. The sudden epidemic events associated with the presence of phytoplasmas molecularly undistinguishable, in very distant geographic areas, on the same herbaceous species, provides further indications of transmission of these prokaryotes also by seeds. In all the analysed species, the phytoplasma detected in seedlings belong to different ribosomal groups, in some cases they are found also in mixed infection. Since phytoplasma presence is not contemplated in propagation material by the any of the worldwide plant protection quarantine protocols nor by seed producers, the movement of seeds from infected plants implies a wide dissemination of the pathogens and therefore of the associated diseases in still uncontaminated areas.

A vine decline of kiwifruit (A. deliciosa ‘Hayward’) was observed in 2012 and 2013 in about 600 ha in the province of Verona (Northern Italy). During the last two years, a progressive vine decline took place during summers with high temperatures (over 35°C) requiring copious furrow irrigation, and with mild temperatures and abundant rainfall during winter and spring that caused long periods of soil waterlogging. The diseased plants died after a gradual blight of the leaves. Rotting of the roots intermediate in diameter and of the distal rootlets were associated with all cases of decline. Phytophthora and Pythium species were isolated from the decayed roots, were plate purified and identified by sequence analysis of the internal transcribed spacer (ITS) region of the rDNA amplified with specific primers. Pathogenicity of the Phytophthora isolates was verified with experimental inoculations of kiwi plantlets. Typical symptoms of the decline in the leaves were caused by isolates of Phytophthora cryptogea. This is the first report of Phytophthora and Pythium presence in plants with root rot and vine decline of kiwifruit in Northern Italy associated to anomalous climate condition.

Two genera of the Fabaceae family showed phytoplasmas symptoms in different locations in Europe i.e. Germany and Italy. Spartium junceum L. (Spanish broom) is a deciduous shrub with dark green, round stems and alternate leaves; inflorescences are terminal clusters of several bright yellow somewhat fragrant flowers. This ornamental shrub is frequently spontaneously growing especially in southern Italy where it is affected by spartium witches’ broom (SpaWB) disease, characterized by proliferation of axillary buds and stem fasciation. Two different phytoplasmas have been associated to SpaWB: ‘Candidatus Phytoplasma spartii’ (group 16SrX-D) and a phytoplasma belonging to elm yellows group (16SrV-C). Both were reported associated with SpaWB in Italy while only ‘Ca. P. spartii’ was reported in Spain. In the spring of 2011, typical SpaWB symptoms were observed in a plant up to 2 m tall growing in the city of Ercolano (Campania region, Italy). A similar symptomatology was observed in a group of shrubs of Cytisus scoparius (L) syn. Sarothamnus scoparius, better known as common broom or scotch broom growing in Dahlem botanical garden in Berlin (Germany). This is a perennial shrub native to western and central Europe, but it is considered invasive plant in areas such as North America and New Zealand. Symptomatic and asymptomatic samples were collected in both cases: five samples of C. scoparius and two of S. junceum were analysed for phytoplasma presence by nested-PCR assays employing primer pairs P1A/P7A followed by F1/B6 and R16F2n/R2, phytoplasma identification was achieved by RFLP analyses with Tru1I on the two latter amplicons. Further confirmation of phytoplasma identity was achieved by nested-PCR assays with primers specific for phytoplasma groups 16SrI, 16SrV and 16SrX. All symptomatic samples produced amplicons of the expected lengths and no product was amplified from asymptomatic plants and using 16SrV specific primers. Identification and classification of phytoplasmas allow to detect ‘Ca. P. spartii’ subgroup 16SrX-D and ‘Ca. P. asteris’ subgroup 16SrI-B in both genera. In some of the samples of C. scoparius also stolbur phytoplasmas were identified. Further phytoplasma characterization was carried out on tuf gene using a cocktail primers mix that was able to amplify phytoplasmas identified as ‘Ca. P. asteris’ in S. junceum and phytoplasmas showing two different Tru1I profiles in C. scoparius from Germany that are not present in any published RFLP profile on this gene. Direct amplicon sequencing is in progress in order to verify possible affiliation to ‘Ca. P. spartii’ group since the only available sequences of this gene on phytoplasmas are deposited in Qbank since they were obtained from the Qbol EU project.

Analysis on commercial carrot seeds and derived plants were performed to evaluate the ‘Candidatus Liberibacter solanacearum’ and ‘Candidatus Phytoplasma’ presence. The analyses on the seeds demonstrated the presence of both prokaryotes in nearly 100% of the seed batches tested. Sequencing confirmed that ‘Ca. L. solanacearum’ strains detected in the seed samples belong to the haplotype D. Seedlings were grown in an insect proof greenhouse and in a growing chamber under controlled conditions. Symptoms of root malformation and leaf whitening and browning were observed in some of the carrot plants obtained from seed. DNA extraction and PCR assays were performed on both symptomatic and asymptomatic samples and diverse ‘Candidatus Phytoplasma’ species were detected in the seedlings at different growth stages. DNA belonging to the ribosomal groups 16SrI and 16SrXII-A was detected and confirmed by RFLP analysis and sequencing indicating the seed transmission of these phytoplasmas in carrot. The analyses to detect ‘Ca. L. solanacearum’ in these seedlings resulted always negative.

An island wide survey carried over 18 months across 79 tomato (Lycopersicum esculentum L.) plantations in 30 localities on 12 varieties revealed that symptoms related to phytoplasma-associated diseases are widespread in this species in Mauritius. Typical witches, broom and stunting and/or yellowing were observed in 74.6 % of fields visited. Phytoplasmas belonging to three groups: stolbur (16SrXII), aster yellows (16SrI) and elm yellows (16SrV) were identified in tomato leaf and fruit samples by nested-PCR assays followed by RFLP and/or sequencing analyses. Mixed phytoplasma and virus infections (PVY, TYLCV) were detected in 28.8 % of symptomatic sample tested. Survey results indicate a widespread distribution of all identified phytoplasmas across local tomato plantations (pathogens were detected in 8 out of 9 districts) irrespective of the variety grown. Use of insect-proof nets for seedling production, avoid re-planting a new tomato crop in the vicinity of phytoplasma infected ones and uprooting of symptomatic plants are the recommended measures to reduce the pathogen dissemination and resulted to be effective in reducing the pathogen presence in the Country.

An extensive survey was performed from 2002 to 2006 to detect and identify phytoplasmas associated with Chilean grapevines. Nested polymerase chain reaction assays using phytoplasma universal primer pairs P1/P7 and R16F2n/R2 detected phytoplasmas in 34 out of the 94 samples tested (36%). Restriction fragment length polymorphism (RFLP) analyses, cloning, and sequencing allowed identification of phytoplasmas belonging to ribosomal subgroups 16SrI-B, 16SrI-C, 16SrVII-A, and 16SrXII-A. The 16SrVII-A phytoplasma represents a new finding in grapevine; moreover, variability of the RFLP profile was observed in some of the 16SrXII-A phytoplasmas, indicating possible new ribosomal subgroups. Mixed phytoplasma infections and infections of phytoplasmas together with one or more viruses also occurred.

During 2003–2005 extensive surveys were carried out in diverse location in Serbia to verify phytoplasma presence in fruit trees species affected by decline, reddening, yellowing or witches’ broom symptoms. Tests were carried out using PCR/RFLP analyses to detect phytoplasma presence and to determine their identity. European stone fruit yellows phytoplasmas (16SrX-B) were identified in apricot plants showing typical chlorotic leaf roll symptoms and in samples collected from peach plants showing general decline together with yellowing and reddening of leaves. In other samples from peaches with similar symptoms, stolbur (16SrXII-A) phytoplasmas were detected. Two different symptoms were associated with diverse phytoplasmas in pear: pear decline (16SrX-C) phytoplasmas were identified in samples from plants showing typical decline symptoms, while X-disease (16SrIII-B) phytoplasmas were identified in a pear plant showing witches’ broom and reduced growth. Samples from apple showing symptoms of proliferation, witches’ broom and reduced fruit size, resulted to be infected by apple proliferation (16SrX-A) phytoplasmas.

Erysimum linifolium L. (sin. Cheiranthus linifolium L.) (Brassicaceae), or Aegean wallflower, native to the Mediterranean region, is an evergreen perennial compact shrub that offers silvery-green foliage and beautiful spikes of lilac-mauve flowers through long periods, starting in mid-spring. In Liguria region (northern Italy) this species is mostly cultivated to be used in rock gardens or in mixed garden borders. In 2012, a phytoplasma-like disease was observed in a few pot-plants at an ornamental grower of Albenga area (Liguria region). Symptomatic E. linifolium showed reduced leaf size, rosetting and stunting; in some cases, shortening of internodes and growing reduction involved only a part of the plant. After first symptoms observation, an increasing percentage of symptomatic plants were found at flowering stage, when affected plants did not bloom. In order to verify phytoplasma presence and to determine their identity, samples from symptomatic and asymptomatic plants were collected and tested, after a chloroform/phenol nucleic acid extraction by direct PCR with primers P1A/P7A followed by nested-PCR with primers F1/B6 and R16F2n/R2. RFLP analyses performed with Tru1I and HhaI enzymes, allowed the identification, only in the symptomatic plants, of phytoplasmas belonging to subgroup 16SrI-B (‘Candidatus phytoplasma asteris’). Further confirmation of phytoplasmas identity was obtained after Tru1I RFLP analyses on tuf gene amplified with cocktail primers. ‘Candidatus Phytoplasma asteris” (16SrI) is associated with over 100 economically important plant diseases and represent on of the most diverse and widespread phytoplasma groups. Strains that belong to subgroups 16SrI-A, 16SrI-B and 16SrI-C are distributed worldwide and are associated with diseases in more than 80 plant herbaceous species transmitted by more than 30 species of insect vectors. The 16SrI-B represents the largest and most diverse strain cluster in the group in which at least 20 ribosomal subgroups were recognized. Some of the subgroups were only detected in woody hosts such as 16SrI-P identified in poplar in Eastern Europe, 16SrI-S and 16SrI-Q detected in cherry respectively in China and in Lithuania indicating the ability of some of its strains to infect all kind of plant species. The detection and identification of aster yellows phytoplasmas in E. linifolium in Italy represents however its first report in this species worldwide. Phytoplasmas belonging to 16SrII group (‘Ca. P. aurantifolia’) were detected in 2010 in E. cheiri (sin. C. cheiri) a different species cultivated in south-eastern Iran. In this case, infected plants showed witches’ broom and phyllody. Considering that E. linifolium is propagated by seed, it is very likely that leafhoppers are involved in 16SrI-B phytoplasma spreading in this species. Work on possible insect vector identification is in progress.

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.

Nonostante negli ultimi anni la fl avescenza dorata sia tornata a manifestarsi in alcuni areali, la situazione nella zona del Conegliano Valdobbiadene docg resta sotto controllo non provocando rilevanti perdite di produzione

Da anni l'Area di patologia vegetale sostiene le cooperative liguri specializzate nella produzione di piante ornamentali, orticole ed officinali per quanto riguarda la difesa dalle malattie dovute a fitoplasmi

Turnera ulmifolia, the yellow alder, is a widely distributed species in Brazil where, besides ornamental, it is used for its medicinal properties. Plants of T. ulmifolia exhibiting witches’ broom growths and yellowing, that are symptoms typically associated with phytoplasma presence, have been observed in the location of Penedo, state of Rio de Janeiro. Symptomatic samples from T. ulmifolia were collected and after total nucleic acid extraction, universal primer pairs were used to prime amplification of phytoplasma 16S rDNA sequences, spacer region and beginning of the 23S rDNA. Expected length amplicons of 1.5 kb (F1/B6 primers) and 1.2 kb (R16F2/R2 primers) were obtained from all symptomatic samples tested after nested PCR on P1/P7 amplicons. RFLP analyses were carried out with Tru1I on F1/B6 and R16F2/R2 amplicons; and with TaqI, and AluI on R16F2/R2 amplicons. RFLP patterns were compared with those of phytoplasma reference strains on same size amplicons and allow to identify the detected phytoplasmas as belonging to ribosomal group 16SrXIII. Additional amplifications for molecular characterization of T. ulmifolia phytoplasmas, with primers rp(I)F1/rp(I)R1A amplifying rplV (rpl22) and rpsC (rps3) genes were then performed. The amplification of this gene resulted in the expected 1.2 kb amplicons and the RFLP profile obtained after Tru1I digestion was clearly different from any of those available in the literature for the same gene, indicating that this phytoplasma may represent a new strain in 16SrXIII group.

Erigeron bonariensis, the flax-leaved fleabane is found in several regions of Brazil. In the State of Rio de Janeiro, plants of E. bonariensis exhibiting shortened internodes, reduced leaf size, witches’ broom and yellowing were tested for phytoplasma presence. Amplicons were obtained from nested PCR with primers designed for 16S rRNA and tuf genes. RFLP analyses of the ribosomal DNA revealed similarity to phytoplasmas of the ash yellows group and sequence analyses demonstrated 100% identity with phytoplasma strains belonging to subgroup 16SrVII-B from Argentina and Brazil. The sequence from tuf gene showed a maximum of 94% identity with ‘Candidatus Phytoplasma fraxini’ strain ASY3, and 92% identity with other strains of the ash yellows group. To our knowledge, this is the first report of the species Erigeron bonariensis harboring a ‘Ca. P. fraxini’-related strain in Brazil.

In contrast to mycoplasmas, which cause an array of disorders in animals and humans, phytoplasmas resisted all attempts to culture them in cell-free media. Despite reduced genome size in comparison to their ancestors, they retain an independent metabolism that allows them to survive in environments as diverse as plant phloem and insect haemolymph. This versatility is a unique property among microbes, shared only with some animal- or plant-infecting viruses and a few other microorganisms such as the causal agent of malaria. Very recently the proof that phytoplasmas can now be grown on laboratory media was provided employing specific commercially available media. Several key points were important to achieve this result: plant tissue selected for isolation must contain viable phytoplasmas; release of phytoplasmas must be achieved from the sieve-tubes in the presence of sieve-tube sap coagulation mechanisms to prevent loss on trauma, the incubation period must be of sufficient time to allow evidence of phytoplasma growth to manifest itself. Inadequacy on any of these points would result in failure so there are many reasons, apart from a suitable medium, that could be responsible for previous failures. Besides the initial seven phytoplasmas, six additional strains were grown: ‘Candidatus Phytoplasma trifolii’ (strain PWB, ribosomal group 16SrVI-A), ‘Ca. P. aurantifolia’ (strain WBDL, ribosomal group 16SrII-B), ‘Ca. P. pruni’ (strain CX, ribosomal group 16SrIII-A), ‘Ca. P asteris’ (strain KVE, ribosomal group 16SrI-C), ‘Ca. P. phoenicium’ (strain PEY, ribosomal group 16SrIX-C), and ‘Ca. P. prunorum’ (strain PLNV6, ribosomal group 16SrX-B). Additional types of phytoplasmas are in the initial isolation stage, but it appears that all the available phytoplasma strains can be cultured. Contrary to the prevailing dogma in plant pathology, therefore, phytoplasmas, like mycoplasmas, can indeed be grown independently from the host(s). The results will produce a more detailed knowledge about basic mechanisms that regulate the survival of phytoplasmas, which are among the smallest known living organisms, but can induce among the most severe epidemics in agriculture.

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.

Bermuda grass white leaf (BGWL) is a destructive disease of bermuda grass (Cynodon dactylon L.) associated with the presence of ‘Candidatus Phytoplasma cynodontis’. Strains belonging to 16SrXIV bermuda grass white leaf group were detected in several tropical and subtropical areas worldwide and regions bordering to these climates. BGWL strains can be separated by genetic markers from economical important phylogenetic-related strains such as sugarcane white leaf (SCWL), sugarcane grassy shoot (SCGS), rice yellow dwarf (RYD) and sorghum grassy shoot (SGS) phytoplasmas. Beside this interesting relationship, BGWL was described carry the smallest chromosome in phytoplasmas. Little is known about the genomes of BGWL strains and the coding of effectors or membrane proteins probable interacting with host and/or vector. In a genomic survey, a first draft sequence of a BGWL from Italy was determined. Preliminary analysis of the gene content highlighted the coding of an imp-gene fragment. The imp-region of three samples from Italy was reconstructed by a PCR-approach using four primers derived from the genomic draft. The impregion of these samples, above 5 kb in size, encodes a dsRNA-specific ribonuclease RNaseIII (Rnc), a chromosome replication initiation protein (DnaD), imp-like protein (Imp), a CTP synthase (PyrG), probable a phosphatidylserine decarboxylase (psd) and a phosphatidylserine synthase (PssA). Gene order of this region is identical to ‘Candidatus Phytoplasma oryzae’ (acc. no. AB469012) derived from a Japanese strain detected in Farfugium japonicum var. Kitamura. Partial sequences of the 5’- end of imp were determined for six samples showing no variation in their nucleotide sequence to each other indicating an unexpected conservation of this gene encoding a putative membrane protein in BGWL strains.

The importance of diagnostic procedures harmonization, the contribution to improved transparency during the detection of regulated pests and the need of the resolution of disputes among trading partners suggested, in the frame of the Italian Project ARNADIA financed by the Ministry of Agriculture, to set up validated diagnostic protocols, officially approved and published at national level. To select a reference protocol for ‘Candidatus Phytoplasma mali’ and ‘Candidatus Phytoplasma prunorum’ detection different diagnostic methods, protocols and reagents have been compared in five laboratories, using the same target and non-target reference samples. Methods of conventional and real time PCR have been selected and developed to determine the performance characteristics for the validation under the standard ISO 17025. The results show that the accuracy and sensitivity of conventional and real time PCR are comparable, whereas real time RT-PCR is recommendable to increase the analytical sensitivity. The reference protocols are available under www.strateco.it.

Kentia (Howea forsteriana) is worldwide grown as ornamental especially for inside decorations. Phytoplasmas have been associated with several diseases of palm trees with symptoms that are quite variable and consist of leaf yellowing, reduction in fruit and stalk size, stunting, wilting and severe or slow decline. Lethal yellowing, lethal decline, root wilt, white tip die-back and Al-Wijaam are some of the reported phytoplasmas-associated diseases associated with different species of palm in the world. Lethal yellowing is however a disease associated worldwide with phytoplasmas that affects at least 30 species of palm, among which the most relevant are Phoenix dactylifera, Veitchia merrilli, Caryota rumphiana, Phoenix canariensis and Elaeis guineensis. This disease has killed millions of palm trees such as coconut (Cocos nucifera) throughout the Caribbean, Florida, Mexico, and the Central American region. Kentia plants showing severe symptoms of decline quite similar to the lethal yellowing were observed in outside garden in some areas of Eastern Sicily. To study the disease ten potted plants were collected a few months after sprouting and maintained under insect-proof greenhouse. Total nucleic acids were extracted from 1 g of tissue from leaves and petioles with a chloroform/phenol-based method and direct and nested PCR assays were carried out in two different period of the year (Autumn and Spring). All the samples were negative to phytoplasma presence while in some cases RFLP profiles and sequences related to possible endophitic bacteria were detected. A second nested PCR assay with more specific primers allow to detect phytoplasmas that were identified as belonging to aster yellows (16SrI-B) and “stolbur” (16SrXII-A) ribosomal groups. The detection of these phytoplasmas in kentia with decline symptoms increases the number of plant species that can be infected by these prokaryotes. It is however necessary to further evaluate the possible presence of other microorganisms that may contribute to determine the severe disease detected in open field in order to devise the appropriate disease management to contain its spread.