Identification and GroEL gene characterization of green petal phytoplasma infecting strawberry in Italy

none5The presence of phytoplasmas in strawberry showing malformation of the fruits together with the typical green petals symptoms was detected in some North Western Italy cultivations. Nucleic acids extracted from these plants were used in nested-PCR assays with primers amplifying 16S rDNA and GroEL sequences specific for phytoplasmas. Bands of 1.2 kb were obtained in both cases after nested-PCR assays and RFLP analyses allowed to classify the detected phytoplasmas in the aster yellows subgroup 16SrI-C, the GroELI grouping confirm all the strains from strawberry to be identical to each other and to GroELI-VI group. This is the first multigene molecular identification of strawberry green petals phytoplasmas in Italy.openContaldo N.; J.F. Mejia; S. Paltrinieri; A. Calari; A. BertacciniContaldo N.; J.F. Mejia; S. Paltrinieri; A. Calari; A. Bertaccin

Axenic culture of plant pathogenic phytoplasmas

Phytoplasmas are microorganisms associated with severe plant diseases affecting diverse agrarian activites worldwide. Since their discovery, conclusive proof of their pathogenicity is lacking due to failure to culture these organisms. This has also hindered studies on their biology, biochemistry and physiology, although significant taxonomic progress has been achieved from study of the 16S ribosomal gene and full genomic sequencing of four phytoplasma strains. The inability to fulfil Koch’s postulates severely restricts the understanding of the roles of these organisms in plant diseases and in plant/insect/phytoplasma interactions. Here we show that specific commercial media support axenic growth of phytoplasmas under defined conditions; the identity of the organisms was confirmed by PCR/RFLP analyses and sequencing of phytoplasma-specific genes. We have demonstrated for the first time that phytoplasmas, similarly to mycoplasmas, can grow independently from their host(s). This should assist reduction of the socio-economic impact of phytoplasma diseases worldwide through improved pathogen detection and consequently better management of the diseases they cause. Knowledge of mechanisms underlying the autonomous life of phytoplasmas, that are among the smallest living organisms, should also provide important information about basic mechanisms of life

Simultaneous detection of mixed ‘Candidatus Phytoplasma asteris’ and ‘Ca. Liberibacter solanacearum’ infection in carrot

Symptoms of shoot and root malformation were observed in carrot plants in fields located in the North of Gran Canaria Island (Spain), during surveys carried out in spring 2015 and 2016. Total DNA extraction from the leaves of symptomatic plants was performed and PCR assays were carried out to detect the agents possibly associated to the observed symptoms. While ‘Candidatus Liberibacter solanacearum’ was detected in the majority of tested symptomatic samples, phytoplasmas belonging to the ribosomal group 16SrI were detected only in two samples. Phylogenetic analyses and sequencing, together with virtual RFLP, confirmed that ‘Ca. L. solanacearum’ strains detected in carrot samples belong to haplotype D

Axenic culture of plant pathogenic phytoplasmas

open5siPhytoplasmas are microorganisms associated with severe plant diseases affecting diverse agrarian activites worldwide. Since their discovery, conclusive proof of their pathogenicity is lacking due to failure to culture these organisms. This has also hindered studies on their biology, biochemistry and physiology, although significant taxonomic progress has been achieved from study of the 16S ribosomal gene and full genomic sequencing of four phytoplasma strains. The inability to fulfil Koch’s postulates severely restricts the understanding of the roles of these organisms in plant diseases and in plant/insect/phytoplasma interactions. Here we show that specific commercial media support axenic growth of phytoplasmas under defined conditions; the identity of the organisms was confirmed by PCR/RFLP analyses and sequencing of phytoplasma-specific genes. We have demonstrated for the first time that phytoplasmas, similarly to mycoplasmas, can grow independently from their host(s). This should assist reduction of the socio-economic impact of phytoplasma diseases worldwide through improved pathogen detection and consequently better management of the diseases they cause. Knowledge of mechanisms underlying the autonomous life of phytoplasmas, that are among the smallest living organisms, should also provide important information about basic mechanisms of life.openContaldo N.; A. Bertaccini; S. Paltrinieri; H.M. Windsor; D.G. WindsorContaldo N.; A. Bertaccini; S. Paltrinieri; H.M. Windsor; D.G. Windso

Molecular characterisation of bois noir phytoplasmas from Bosnia and Herzegovina

Bois noir (BN) phytoplasmas were associated with grapevine yellows diseases in Bosnia and Herzegovina (Delic and Lolic, 2010). The disease is widely to moderate distributed in vineyards with both domestic and imported grapevine cultivars. Molecular characterisation of selected strains of BN phytoplasmas detected in heavily BN-infected vineyards in was done to verify the presence of genetic variability. Thirty-five DNA samples of grapevines that were shown to be positive by PCR assays on the 16S rDNA were selected for the study. Polymorphisms were studied in the 16S rDNA, Tuf and ribosomal protein genes of the selected samples. Tuf1f/r, TufAYf/r, and TufINT1f/TufINT4r primer pairs were used in nested PCR for amplification of Tuf genes; RFLP analyses with HpaII showed the presence of the tuf type-b (VK-II) in all the tested samples. The BN infected samples were further amplified with rp/STOL primers and PCR products were digested with TaiI and SspI restriction enzymes. Finally 16S rDNA genes were amplified with P1/P7 primers in direct PCR and with R16F2n/R16R2 primers in nested PCR assays: both types of amplicons obtained were restricted with MboII and RsaI, MboII, Hpy188I enzymes, respectively. Restriction profiles obtained after RFLP analyses on 16S rDNA and ribosomal protein amplicons weren't uniform. Some samples showed profiles comparable with some of those recently published (Contaldo et al., 2009), while some of the profiles of other samples were totally different from any of those reported in literature. Therefore, polymorphism of these regions should be further studied to better understand BN epidemiology in Bosnia and Herzegovina

Phytoplasmas and Phytoplasma Diseases: A Severe Threat to 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

Biological and molecular proof of phytoplasma seed transmission in corn

Phytoplasma seed transmission is still a poorly investigated topic inspite of growing experimental evidences. Corn seeds deriving from 5 symptomatic and phytoplasma-infected cobs were sown and the germinated plants were analyzed to verify the presence of infected progenies by nested PCR on 16S ribosomal DNA, and by attempts of phytoplasma cultivation in chemically defined media to verify their viability. After sterilization, seeds were in vitro germinated and the plantlets were transferred in sterile soil in insect-proof cages and tested when they were 30 to 100 days old. Preliminary trials were carried out on 9 seedlings, 6 of which, positive to aster yellows and “stolbur” phytoplasmas, were used as plant sources for isolation trials in PivL® medium. From three plants the isolation allowed to detect aster yellows and “stolbur” phytoplasma DNAs after chloroform/phenol extraction from 1 ml of liquid medium from isolation tubes and from tubes obtained after serial dilution that have shown color change. Further tests on 79 seedlings resulted in 17 of them positive for aster yellows and “stolbur” phytoplasmas. Isolation from three of these plants after 30 days from germination resulted positive for phytoplasma DNA in tubes deriving after 2 to 10 serial dilution in fresh medium that have all shown colour change.Reisolation carried out at 90 days from germination confirmed these results only from one plants. Plating carried out together with DNAs extraction from liquid medium produced colonies of different size and shapes. The same type of colonies were obtained from plating tubes maintained for seven month at 25°C. Single colonies were picked and transferred in broth for purification steps at several times and small colonies were obtained resulting to consistently contain aster yellows DNAs; moreover this type of colony growth was observed for at least 3 subsequent passages liquid/solid media carried out about every 5 days. These preliminary results are indicating the viability of phytoplasmas isolated in corn seedlings grown in insect-proof environment

STRATEGIA DI DIFESA FITOSANITARIA DELLE SPECIE OFFICINALI PER UNA MAGGIORE VALORIZZAZIONE DELLA PRODUZIONE ERBORISTICA LEGATA AL TERRITORIO

I maggiori produttori di piante officinali sono in Europa sono Ungheria, Polonia, Bulgaria, Francia, Belgio ed Olanda. Negli ultimi 15 anni anche in Italia \ue8 aumentata sensibilmente la richiesta di piante officinali da parte di industrie farmaceutiche, cosmetiche ed erboristerie a seguito di una maggiore consapevolezza ed attenzione del \u201cconsumatore\u201d nei confronti di sostanze naturali e prodotti biologici. La crescente richiesta di specie officinali (medicinali ed aromatiche) ha determinato un aumento sensibile delle superfici coltivate, insufficiente per\uf2 a soddisfare la domanda interna. Continua quindi ad essere necessario importare materiali vegetali (piante intere e/o parti di pianta) che, pur essendo molto competitivi dal punto di vista economico, sono purtroppo nettamente inferiori a quelli nazionali in termini di qualit\ue0 sanitaria. La produttivit\ue0 degli impianti e la qualit\ue0 delle droghe vegetali sono, infatti, spesso minacciate da fitopatie epidemiche, quali fitoplasmosi e virosi, in grado di compromettere la resa degli impianti, cos\uec come di modificare qualitativamente e quantitativamente i principi attivi che costituiscono la droga. Il risultato \ue8 un prodotto di qualit\ue0 scadente. L\u2019aspetto sanitario delle coltivazioni occupa quindi un ruolo importante nell\u2019economicit\ue0 degli impianti e nella standardizzazione del prodotto finale destinato al consumo diretto od alla trasformazione industriale. Da qui l\u2019interesse nel valutare l\u2019incidenza delle malattie dovute a virus ed a fitoplasmi in coltivazioni di piante medicinali ed officinali site nel Comune di Sogliano sul Rubicone (Forl\uec-Cesena), zona collinare sui crinali dell\u2019Appennino Tosco-Marchigiano a 379 metri s.l.m. con lo scopo di indicare la profilassi da adottare per prevenire l\u2019insorgere di queste malattie e valorizzare nostra la produzione erboristica. Durante numerosi sopralluoghi eseguiti nel triennio 2004-2007, sono stati ispezionati diversi appezzamenti raccogliendo campioni vegetali (foglie e/o fiori) con sintomi attribuibili alla presenza di virus e/o fitoplasmi. Sono state esaminate le seguenti specie: Hyssopus officinalis L. caratterizzato da giallume fogliare, virescenza e nanismo; Galega officinalis L. con sintomi di giallume e nanismo; Salvia sclarea L. caratterizzata in alcuni casi da mosaico giallo oro sulle foglie e nanismo, in altri da giallume; Parietaria officinalis L. e P. judaica Auct. An. L., entrambe mostranti sintomi di giallume e nanismo. Per verificare la presenza di eventuali infezioni da virus sono state eseguite prove di trasmissione meccanica e saggi immunoenzimatici applicando la tecnica PAS-ELISA. Per quanto riguarda la diagnosi per la presenza di fitoplasmi, sono state applicate le tecniche PCR e RFLP. Le specie esaminate sono risultate infette da fitoplasmi appartenenti prevalentemente al gruppo 16SrXII (stolbur), considerato \u201cubiquitario\u201d sia in Italia che in Europa ed individuato in piante erbacee ed arboree (pomodoro, patata, fruttiferi e vite) ove \ue8 in grado di provocare anche forti epidemie e da 2 virus: BBWV e AMV

Sharing information and collections on phytoplasmas: from QBOL to QBANK

Abstract A total of 154 phytoplasma strains from 15 ribosomal groups were employed for barcode sequences production. Besides strains in periwinkle, 36 strains in natural infected plants such as napier grass, grapevine, plum, jujube, apple, pear, spartium, pine tree, hibiscus and erigeron were employed. Barcode sequences were produced for 16SrDNA, tuf and SecA gene for 36 phytoplasma strains, while for 54 strains 16SrDNA sequences were obtained, and for 118 and 89 strains respectively the tuf and secA barcode. All obtained sequences and protocol for extraction and PCR amplification will be available in Qbank

Phytoplasma detection and identification in grapevine by deep amplicon sequencing

Grapevine yellows is one of the major diseases affecting the vineyards in Europe as well as in the majority of grapevine cultivation areas worldwide. Phytoplasma presence seriously affect quality and quantity of production therefore accurate and sensitive pathogen detection and identification are relevant for the disease management. Phytoplasmas within single plants are populations of individuals; routine techniques such as PCR followed by RFLP or sequencing of uncloned or cloned products do not show the diversity of these populations. These techniques only allow identification of the most frequent genotypes in the samples and not mixed phytoplasma or pathogen infection. A number of grapevine samples in which single or mixed phytoplasma infection was detected by routine methods were employed for deep amplicon sequencing on the Roche Genome Sequencer FLX system to compare the two detection systems. Materials and Methods Samples employed were selected from those that are routinely processed during surveys for phytoplasma detection and identification in Northern Italy where both ‘flavescence dorée’ (FD) and ‘bois noir’ (BN) diseases are widespread or epidemic (Botti and Bertaccini, 2007). Total DNA was extracted from 1 g of mid-vein leaf tissue following the procedure of Angelini et al., (2001). Phytoplasma detection was carried out by direct PCR on ribosomal gene and spacer region (Martini et al., 2002) followed by nested amplification with R16(I)F1/R1 (Lee et al., 1994) and 16R758F/V1730 (Martini et al., 1999) primer pairs. RFLP analyses with TruI on the first amplicons and TaqI on the second one allow identification of BN and FD phytoplasmas. Samples showing single and mixed phytoplasma infection were selected for deep amplicon sequencing. Tagged primers were used for generating pyrosequencing samples following described PCR protocol (Nicolaisen et al., 2011). PCR products were pooled in equimolar amounts, run on an agarose gel and a band of the correct size was excised from the gel and purified using QIAquick gel extraction kit from QIAGEN. Two pools of 13 and 11 samples respectively were sequenced on a GS FLX plate at Eurofins MWG. Tag-sorted sequences were quality filtered using CLOTU software at the Bioportal webportal (http://www.bioportal.uio.no/). To minimize sequencing errors, only the first ~200 nucleotides of each sequence were used. Accepted sequences were clustered using CD-HIT with a 99% similarity threshold, and singleton sequences were discarded. To identify sequences, these were aligned together with reference sequences from GenBank using MEGA and phylogenetic trees were constructed. Results and Discussion A total of 35521 sequences were generated from the first pool of 13 field collected samples of phytoplasma infected grapevine in which single infection was detected by routine procedures. Four of these samples were tested twice to verify result consistency (table 1). These data overall confirmed RFLP results. Table 1. Number of sequences determined in each sample with single phytoplasma infection by routine methods. FD-C 46 FD-C 51 FD-D 53 FD-D 64 FD-D 66 FD-D 54 BN 33 BN 43 BN 59 FD-C 56 FD-C 63 FD-C 68 FD-C 71 FD-C 46 FD-D 53 BN 33 FD-C 56 16SrV-C/D 2109 1709 3032 885 1559 42 19 15 10 2040 1876 1520 1356 2126 2240 0 2540 16SrXII-A 0 0 0 0 24 0 2213 2730 4716 3 1 0 0 0 0 2756 0 Table 2. Number of sequences determined in each sample with mixed phytoplasma infection determined by routine methods. FD+BN 63 FD+BN 71 FD+BN 73 FD+BN 60 FD+BN 72 BN 67 FD+BN 76 FD TV1 FD+BN 112 FD+BN 120 FD+BN 80 16SrV-C/D 3009 414 54 612 359 2158 130 201 914 3846 33 16SrXII-A 361 5447 4442 1424 3964 1019 9727 4574 698 0 6823 16SrX-B 189 0 0 0 0 109 0 0 278 0 0 Chimeras 39 17 16 8 26 0 11 0 24 0 0 Total 3598 5878 4512 2044 4349 3286 9868 4775 1914 3846 6856 A total of 50,926 sequences were generated from the pool of 11 field collected samples in which mixed BN and FD phytoplasma infection was ..