Genetic relatedness and recombination analysis of Allorhizobium vitis strains associated with grapevine crown gall outbreaks in Europe.

AIMS: To analyse genetic diversity and epidemiological relationships among 54 strains of Allorhizobium vitis isolated in Europe during an 8-year period and to assess the relative contribution of mutation and recombination in shaping their diversity. METHODS AND RESULTS: By using random amplified polymorphic DNA (RAPD) PCR, strains studied were distributed into 12 genetic groups. Sequence analysis of dnaK, gyrB and recA housekeeping genes was employed to characterize a representative subcollection of 28 strains. A total of 15 different haplotypes were found. Nucleotide sequence analysis suggested the presence of recombination events in A. vitis, particularly affecting dnaK locus. Although prevalence of mutation over recombination was found, impact of recombination was about two times greater than mutation in the evolution of the housekeeping genes analysed. CONCLUSIONS: The RAPD analysis indicated high degree of genetic diversity among the strains. However, the most abundant RAPD group was composed of 35 strains, which could lead to the conclusion that they share a common origin and were distributed by the movement of infected grapevine planting material as a most common way of crossing long distances. Furthermore, it seems that recombination is acting as an important driving force in the evolution of A. vitis. As no substantial evidence of recombination was detected within recA gene fragment, this phylogenetic marker could be reliable to characterize phylogenetic relationships among A. vitis strains. SIGNIFICANCE AND IMPACT OF THE STUDY: We demonstrated clear epidemiological relationship between majority of strains studied, suggesting a need for more stringent phytosanitary measures in international trade. Moreover, this is the first study to report recombination in A. vitis.

Differentiation and classification of phytoplasmas in the pigeon pea witches'-broom group (16SrIX): an update based on multiple gene sequence analysis.

The pigeon pea witches'-broom phytoplasma group (16SrIX) comprises diverse strains that cause numerous diseases in leguminous trees and herbaceous crops, vegetables, a fruit, a nut tree and a forest tree. At least 14 strains have been reported worldwide. Comparative phylogenetic analyses of the highly conserved 16S rRNA gene and the moderately conserved rplV (rpl22)-rpsC (rps3) and secY genes indicated that the 16SrIX group consists of at least six distinct genetic lineages. Some of these lineages cannot be readily differentiated based on analysis of 16S rRNA gene sequences alone. The relative genetic distances among these closely related lineages were better assessed by including more variable genes [e.g. ribosomal protein (rp) and secY genes]. The present study demonstrated that virtual RFLP analyses using rp and secY gene sequences allowed unambiguous identification of such lineages. A coding system is proposed to designate each distinct rp and secY subgroup in the 16SrIX group.

First Report of 'Candidatus Phytoplasma asteris'-Related Strain Associated with Peach Rosette in Canada.

Prunus persica (L.) Bastch (family Rosaceae) is currently represented by 83 accessions at the Canadian Clonal Genebank. Approximately 3,200 ha are devoted to peach cultivation in Canada where Ontario Province accounts for 82% of the national production. The clonal peach accessions, also located in Ontario, are monitored routinely for symptoms of phytoplasma infection, including rosette-like symptoms (3) that are characterized by new shoots with very short internodes, loss of older shoot leaves leaving only bunches of young leaves on the tips of naked shoots, and flowers that rarely set fruit. From June to August 2009, peach accessions PRU0382 and PRU0445 showed typical peach rosette symptoms, while another 14 accessions exhibited either short internodes or no symptoms. Leaf midrib samples were collected from 16 peach accessions, including 17 symptomatic (from which 8 corresponded to accession PRU0382, 6 for PRU0445, 1 for PRU0335, 1 for PRU0179, and 1 for PRU0451) and 16 asymptomatic (from which 5 corresponded to a representative of each accession PRU0382, PRU0445, PRU0335, PRU0179, and PRU0451 and 11 to other peach accessions). Total DNA was extracted (DNeasy Plant Extraction Mini Kit, QIAGEN, Valencia, CA) from 100 mg of each sample and used as a template in a nested PCR with phytoplasma universal primers R16mF2/R1 (1) and fU5/rU3 (2). Nested PCR products of the expected size (~880 bp) were obtained from all symptomatic samples (14 of 14) of accessions PRU0382 (peach-almond cv. Kando from the Czech Republic) and PRU0445 (peach cv. HW271 from Canada) only. All other plants with or without symptoms yielded no PCR products. Amplicons were purified (Wizard PCR Clean-up, Promega, Madison, WI), cloned in pGEM-T Easy Vector (Promega), and sequenced (Robarts Institute, London, Canada). The resulting 16S rDNA sequences were identical; one of each was archived in GenBank as Accession No. GU223904. BLAST analysis determined that the P. persica phytoplasma sequence shared 99% identity with 16S rDNA sequences of 'Candidatus Phytoplasma asteris'-related strains. This relationship was also supported by restriction fragment length polymorphism analysis (RFLP) of rDNA amplicons using AluI, RsaI, and MseI endonucleases that yielded fragment profiles indicative of phytoplasmas belonging to group 16SrI (Aster Yellows), subgroup B (16SrI-B). Among phytoplasma diseases, those attributed to group 16SrI strains are most numerous and affect the widest plant host range. They include peach rosette in the United States and Europe (3) as well as diseases of various horticultural crops in Canada, including grapevine (4). To our knowledge, this is the first report of a subgroup 16SrI-B phytoplasma affecting peach in Canada. Early detection of phytoplasmas by PCR in accessions with both European and Canadian origins underscores the importance of prompt identification of infected plants for subsequent thermotherapy treatment to maintain the health of the collection and prevent further disease spread. References: (1) D. E Gundersen and I.-M. Lee. Phytopathol. Mediterr. 35:1441, 1996. (2) K. H. Lorenz et al. Phytopathology 85:771, 1995. (3) C. Marcone et al. Acta Hortic. 386:471, 1995. (4) C. Y. Olivier et al. Plant Dis. 93:669, 2009.

A simple and rapid protocol of crude DNA extraction from apple trees for PCR and real-time PCR detection of 'Candidatus Phytoplasma mali'.

Different PCR protocols have been established for detection of European fruit trees phytoplasmas; however the majority of the procedures for extracting phytoplasma DNA are complex, time consuming, and expensive, with a risk of contamination or loss of target DNA. In present study, a crude extract preparation method previously used to detect other plant pathogens was adapted to samples from apple trees infected by 'Candidatus Phytoplasma mali'. End-point and real-time PCR detection of 'Ca. P. mali' were used to compare this extraction procedure with an established method for efficient extraction of purified DNA. The crude extract proved fully adequate for phytoplasma detection in samples from 86 in vitro and 35 in vivo apple shoots or plants and 10 periwinkle plants. High inter- and intra-run reproducibility was obtained for phytoplasma detection with different TaqMan MGB- or SYBR Green-based real-time PCR protocols applied to the crude extracts. Real-time PCR applied to serially diluted crude and purified extracts revealed the same phytoplasma detection limit (dilution up to 10(5)). All results confirm the suitability of this simple, quick, efficient extraction technique for accurate detection of 'Ca. P. mali' in different types of apple and periwinkle samples.

Phytoplasmas Associated with Grapevine Yellows Disease in Chile.

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.

Preliminary evaluation of antimicrobial activity of some chemicals on in vitro apple shoots infected by 'Candidatus Phytoplasma mali'.

Phytoplasmas are associated with several hundred plant diseases worldwide, including numerous ones with important economical impact. Control of epidemic outbreak of phytoplasma diseases can be theoretically carried out by antibiotics. However, they are expensive, not allowed or prohibited in several countries, and even not always efficient. Presently, effective but safe antimicrobial agents are needed to control severe phytoplasma diseases in field. The aim of the present study was to evaluate the susceptibility of 'Candidatus Phytoplasma mali' to several chemical or synthetic antimicrobial agents. We tested nisin, esculetin, pyrithione and chloramphenicol as molecules having different target activities against micro-organisms. Because of their antimicrobial properties against fungi and bacteria, 4 phyto-essential oils (carvacrol, eugenol, terpineol, alpha-pinene) had also been tested. The activity of these molecules was compared with two antibiotics (tetracycline and enrofloxacin) used as control products. All these compounds were tested in in vitro culture of apples (MM106) infected by 'Ca. P. mall'. All compounds were added to the proliferation medium (modified MS) after autoclaving at 3 concentrations (100, 500, 1,000 ppm), except nisin and pyrithione which were tested at 10, 100 and 500 ppm. Phytoplasma infection was quantified in plant materials by real-time PCR before their transfer and after one or two months of culture in the presence of antimicrobial agents. Primary results showed that phytoplasma were not detectable after one and two months in the presence of pyrithione (at 10 and 100 ppm). Moreover, some other products reduced the concentration of phytoplasma after two months. Shoots died or withered on media enriched with essential oils; that made them impossible to assess, especially when they were used at concentration of 500 and 1,000 ppm.

Genetic diversity and vector transmission of phytoplasmas associated with sesame phyllody in Iran.

During 2010-14 surveys in the major sesame growing areas of Fars, Yazd and Isfahan provinces (Iran), genetic diversity and vector transmission of phytoplasmas associated with sesame phyllody were studied. Virtual RFLP, phylogenetic, and DNA homology analyses of partial 16S ribosomal sequences of phytoplasma strains associated with symptomatic plants revealed the presence of phytoplasmas referable to three ribosomal subgroups, 16SrII-D, 16SrVI-A, and 16SrIX-C. The same analyses using 16S rDNA sequences from sesame phyllody-associated phytoplasmas retrieved from GenBank database showed the presence of phytoplasmas clustering with strains in the same subgroups in other Iranian provinces including Bushehr and Khorasan Razavi. Circulifer haematoceps and Orosius albicinctus, known vectors of the disease in Iran, were tested for transmission of the strains identified in this study. C. haematoceps transmitted 16SrII-D, 16SrVI-A, and 16SrIX-C phytoplasmas, while O. albicinctus only transmitted 16SrII-D strains. Based on the results of the present study and considering the reported presence of phytoplasmas belonging to the same ribosomal subgroups in other crops, sesame fields probably play an important role in the epidemiology of other diseases associated with these phytoplasmas in Iran.

Evaluation of tamoxifen and anastrozole in the prevention of gynecomastia and breast pain induced by bicalutamide monotherapy of prostate cancer.

PURPOSE: To determine whether tamoxifen or anastrozole prevents gynecomastia and breast pain caused by bicalutamide (150 mg) without compromising efficacy, safety, or sexual functioning. PATIENTS AND METHODS: A double-blind, placebo-controlled trial was performed in patients with localized, locally advanced, or biochemically recurrent prostate cancer. Patients (N = 114) were randomly assigned to either bicalutamide (150 mg/d) plus placebo or in combination with tamoxifen (20 mg/d) or anastrozole (1 mg/d) for 48 weeks. Gynecomastia, breast pain, prostate-specific antigen (PSA), sexual functioning, and serum levels of hormones were assessed. RESULTS: Gynecomastia developed in 73% of patients in the bicalutamide group, 10% of patients in the bicalutamide-tamoxifen group, and 51% of patients in the bicalutamide-anastrozole group (P < .001); breast pain developed in 39%, 6%, and 27% of patients, respectively (P = .006). Baseline PSA level decreased by > or = 50% in 97%, 97%, and 83% of patients in the bicalutamide, bicalutamide-tamoxifen, and bicalutamide-anastrozole groups, respectively (P = .07); and adverse events were reported in 37%, 35%, and 69% of patients, respectively (P = .004). There were no major differences among treatments in sexual functioning parameters from baseline to month 6. Elevated testosterone levels occurred in each group; however, free testosterone levels remained unchanged in the bicalutamide-tamoxifen group because of increased sex hormone-binding globulin levels. CONCLUSION: Anastrozole did not significantly reduce the incidence of bicalutamide-induced gynecomastia and breast pain. In contrast, tamoxifen was effective, without increasing adverse events, at least in the short-term follow-up. These data support the need for a larger study to determine any effect on mortality.

Apoptosis-related gene expression in benign prostatic hyperplasia and prostate carcinoma.

BACKGROUND: The aim of this study was to examine the expressions of the bcl-2, bax, fas and c-myc apoptosis-related genes in benign prostatic hyperplasia (BPH) and prostate carcinoma (CaP) to determine whether significant differences exist within each disease and between the two groups of patients. The correlation between gene expression and tumour diameter, stage, Gleason score and serum PSA was also investigated. PATIENTS AND METHODS: Tissue specimens from 51 cases of BPH and 27 cases of CaP were examined for bcl-2, bax, fas and c-myc expression by reverse transcriptase-PCR (RT-PCR). RESULTS: In BPH, bcl-2 and bax gave the weakest signals (p < 0.001). In CaP, bcl-2 was the least expressed gene (p < 0.001). In both patient groups, fas and c-myc were the most highly expressed genes (p < 0.05). Both bcl-2 and bax were expressed at higher levels in CaP than in BPH (p < 0.02). The bcl-2/bax ratio was lower in CaP than in BPH (p < 0.001). Bcl-2 was more highly expressed in high Gleason grade (> 7) tumours (p < 0.05). In the BPH group, bax showed a positive relationship with fas (p < 0.01), while the bcl-2 level inversely correlated with that of c-myc (p < 0.05). CONCLUSION: Our data showed that all the apoptosis-related genes were expressed in both BPH and CaP. The stronger expression of bax and the lower bcl-2/bax ratio observed in CaP may suggest a pro-apoptotic stimulus, while the higher bcl-2 levels appear to counterbalance the tendency to cell death.

Influence of bicalutamide with or without tamoxifen or anastrozole on insulin-like growth factor 1 and binding proteins in prostate cancer patients.

BACKGROUND: There is growing evidence that IGF-1 and binding proteins may be involved in prostate cancer promotion and progression. PATIENTS AND METHODS: IGF-1 and binding proteins (IGFBP-1 and 3) serum levels were measured at baseline and after 3 and 6 months of treatment in a selected group of patients with prostate cancer who were randomly assigned to treatment with bicalutamide, bicalutamide plus anastrozole or bicalutamide plus tamoxifen in a comparative study investigating the role of pharmacological medication in the development of bicalutamide-induced gynecomastia. RESULTS: Bicalutamide monotherapy does not appear to alter the IGF-1/IGFBP system. In fact, the increase in IGF-1 levels induced by this treatment was paralleled by comparable increases in binding protein (IGFBP-3). No major changes from baseline up to month 6 either in IGF-1 or in IGFBP-1 and 3 were observed in the bicalutamide plus anastrozole arm. The addition of tamoxifen to bicalutamide produced a sharp decrease in IGF-1 levels (p<0.001) coupled with an increase in both IGFBP-1 (p=0.001) and, to a lesser extent, IGFBP-3 (p=0.5). CONCLUSIONS: The concurrent administration of tamoxifen and bicalutamide reduces the synthesis and bioavailability of IGF-1. Moreover, increased binding protein levels might exert antiproliferative and proapoptotic effects on prostate cancer cells, independently of the IGF-1/IGF receptor-mediated survival system. Both effects might have a synergistic inhibitory influence on prostate cancer growth.

Corn with Symptoms of Reddening: New Host of Stolbur Phytoplasma.

Recurrent epiphytotic outbreaks of a disease of uncertain etiology known as reddening of corn (Zea mays) have occurred in some areas of Serbia during the last 50 years. Affected plants show early and abnormal ripening, dry precociously, and have poor, shriveled grains. Using molecular tools, phytoplasmas were detected in diseased plants and their identity was subsequently deduced as a subgroup 16SrXII-A strain by a variety of supporting assays involving restriction fragment length polymorphism (RFLP) analysis of polymerase chain reaction (PCR)-amplified 16S rDNA and tuf gene sequences, selective amplification of phytoplasma DNA using primer pair G35p/m, similarity of 16-23S intergenic spacer region (SR) sequences, and similarity and phylogenetic analysis of 16S rDNA gene sequences. Presence of stolbur phytoplasmas in corn with reddening symptoms is a new finding not only for Serbia: it is the first report of stolbur phytoplasma in this species worldwide.

First Report of Phytoplasmas in Grapevine in South Africa.

In April 2006, grapevine plants with typical symptoms of yellows (GY) were observed in some South African vineyards. The affected plants showed premature yellowing or reddening and downward rolling of leaves. In some cases, these symptoms were associated with extensive lack of cane lignification that was undistinguishable from yellows symptoms reported in grapevine in the major viticultural areas of the world. Nucleic acids were extracted separately from 0.1 g of fresh leaf midribs and cane phloem scrapes from three symptomatic and three asymptomatic grapevine plants, cv. Shiraz, and from three symptomatic plants, cv. Cabernet, collected from three different locations using Qiagen (Milan, Italy) DNAeasy Plant Mini Kit. A nested polymerase chain reaction (PCR) assay was employed for phytoplasma detection with 2.5 µl of the extracted DNA. Direct and nested PCR assays were performed with P1/P7 (2) and R16F2/R2 (1) universal primer pairs, respectively, obtaining the expected products only from phloem scrapes of the symptomatic plant samples cv. Shiraz. Restriction fragment length polymorphism (RFLP) analyses of R16F2/R2 amplicons with TruI and Tsp509I restriction enzymes, discriminating among phytoplasma ribosomal group and subgroups, showed profiles corresponding to those of "Candidatus Phytoplasma aurantifolia" (ribosomal subgroup 16SrII-B) in all three positive samples. A Stolbur phytoplasma profile (ribosomal subgroup 16SrXII-A) was also observed in one of those samples, indicating the presence of mixed phytoplasma infection (1). Sequencing of the obtained amplicons confirmed the RFLP phytoplasma identification; in particular 16SrXII-A could be the same phytoplasma associated with the 'Bois Noir' disease reported in grapevine; the 1601-bp sequence of 16SrII-B phytoplasma showed 98% similarity to U15442, i.e., to the phytoplasma associated with lime witches'-broom disease in Oman ("Ca. P. aurantifolia") confirming RFLP results. To our knowledge, this is the first report of phytoplasmas in grapevine in South Africa. References: (1) I.-M. Lee et al. Phytopathology 85:728, 1995. (2) B. Schneider et al. Pages 369-380 in: Molecular and Diagnostic Procedures in Mycoplasmology Vol. I. Academic Press Inc., 1995.

Identification of a 16SrII-E Phytoplasma in Calendula arvensis, Solanum nigrum, and Chenopodium spp.

Epidemiological surveys were performed in Northern Sardinia (Italy) in a 10-year-old vineyard affected by "Bois noir" disease. Samples collected between May and October 2003 from chlorotic and stunted weeds belonging to 14 different taxonomic groups were indexed molecularly for detection of phytoplasmas. Nested polymerase chain reaction (PCR) assays using primers specific for the phytoplasma 16SrDNA gene showed three of six Calendula arvensis, one of two Solanum nigrum, and one of seven Chenopodium spp. assayed positive. Restriction fragment length polymorphism analyses and sequencing of amplified 16SrDNA fragments identified a putative phytoplasma in the ribosomal subgroup 16SrII-E. Further characterization of the rps3 gene, coding a ribosomal protein, confirmed the identification. However, the weeds and leafhop-per species collected in the vineyard tested negative by PCR assays for the Stolbur phytoplasma, the causal agent of "Bois noir". This is the first report of a phytoplasma of the 16SrII-E subgroup infecting C. arvensis, S. nigrum, and Chenopodium spp.

Development and evaluation of different complex media for phytoplasma isolation and growth.

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.

Norethisterone enanthate plus testosterone undecanoate for male contraception: effects of various injection intervals on spermatogenesis, reproductive hormones, testis, and prostate.

The goal of this study was to find the most favorable injection interval of norethisterone enanthate (NETE) plus testosterone undecanoate (TU) in terms of gonadotropin, sperm suppression, and prostatic effects. Fifty normal men were randomly assigned to receive NETE 200 mg plus TU 1000 mg every 8 wk (n = 10), every 12 wk (n = 10), every 6 wk for 12 wk and then every 12 wk (n = 10), and every 6 wk for 12 wk and thereafter TU 1000 mg plus placebo every 12 wk (n = 10), and placebo plus placebo every 6 wk for 12 wk and then every 12 wk (n = 10) for 48 wk. Semen analyses, blood drawings, physical examinations, and prostate ultrasounds were performed throughout the study. Of the men in the 8-wk injection group, 90% (nine of 10) achieved azoospermia, compared with 37.5% (three of eight) in the 12-wk injection group (P = 0.019). TU plus placebo injected every 12 wk did not maintain sperm suppression. Prostate volumes did not change significantly in either group. In conclusion, these data suggest that the combined administration of NETE and TU at 8-wk intervals represents an effective hormonal contraceptive regimen.

Home and office uroflowmetry for evaluation of LUTS from benign prostatic enlargement.

A group of 107 patients with lower urinary tract symptoms (LUTS) from benign prostatic enlargement (BPE) participated to the HOUSE Study (Home and Office Uroflowmetry Specific Evaluation). Patients received routine investigation, consisting of medical history taking, physical examination including digital rectal examination, prostate-specific antigen (PSA), assessment of symptoms listed both on the International Prostate Symptom Score and on ICS-male questionnaire. We examined the results of uroflowmetry evaluation in this population; data were analysed to observe if any circadian changes of parameters obtained with home uroflowmetry could be detected. We searched a correlation between Q(max), Q(ave) and ICS-benign prostatic hyperplasia symptom score: a significantly inverse correlation was found only for Q(max), confirming Q(max) as a reliable parameter to quantify subjective symptoms. When examining the multiple flow curves recorded in the same patient with home uroflowmetry, voided volume and flow time had usually higher values during night-time: the existence of circadian changes of uroflowmetry parameters in patients with LUTS from BPE was confirmed, and lower values of average and maximum flow rates during sleep hours were recorded in the same patient. In conclusion, when evaluating the natural history or treatment outcome of individual patients or group of patients in clinical trials for evaluation of BPE and LUTS, an assessment including multiple measurements may be useful and of value.

First Report of Pear Decline Phytoplasmas on Pear in Serbia.

During August of 2004, pear (Pyrus communis L.) plants with typical symptoms of pear decline (PD) were observed in orchards in central Serbia. The affected plants showed premature reddening and upward rolling of leaves that often showed down-turned petioles. In some cases, premature defoliation was observed. Although a similar decline of pear was observed earlier, until now, the causal agent had not been identified. DNA was extracted with a chloroform/phenol procedure from fresh leaf midribs and branch phloem scrapes of four symptomatic and one asymptomatic pear plants separately. A nested polymerase chain reaction assay (PCR) was used for phytoplasma detection (first PCR round with P1/P7 (4) phytoplasma universal primer pair, followed by nested PCR with group 16SrX specific primers f01/r01) (3). With these primers, the expected products from phloem scrapes and midrib extracts of symptomatic plant samples were obtained. Restriction fragment length polymorphism (RFLP) analyses of the f01/r01 amplicon, with RsaI and SspI restriction enzymes, discriminating among 16SrX subgroup phytoplasmas, showed profiles corresponding to those of the apple proliferation phytoplasma group, 16SrX-C subgroup, "Candidatus Phytoplasma pyri" (2). A 1,155-bp sequence of 16S rDNA gene for one of the PA2f/r (1) amplicons obtained in nested PCR on P1/P7 products from one of the leaf midrib samples was deposited in GenBank (Accession No. AY949984); both strands of the fragment were sequenced with the Big Dye Terminator reaction kit (Applied Biosystems, Foster City, CA). The sequences were analyzed with the Chromas 1.55 DNA sequencing software (Technelysium, Queensland, Australia) and aligned with BLAST software ( http://www.ncbi.nlm.nih.gov ). The blast search showed 100% homology of this sequence with that of PD strain Y16392, confirming the identity with PD of the phytoplasma detected. To our knowledge, this is the first report of pear decline phytoplasmas in Serbia. References: (1) M. Heinrich et al. Plant Mol. Biol. Rep. 19:169, 2001. (2) IRPCM Phytoplasma/Spiroplasma Working Team-Phytoplasma Taxonomy Group. Int. J. Syst. Evol. Microbiol. 54:1243, 2004. (3) K.-H. Lorenz et al. Phytopathology 85:771, 1995. (4) Schneider et al. Pages 369-380 in: Molecular and Diagnostic Procedures in Mycoplasmology. Vol I. S. Razin and J. G. Tully, eds. The American Phytopathological Society, 1995.

Testosterone undecanoate maintains spermatogenic suppression induced by cyproterone acetate plus testosterone undecanoate in normal men.

In this study we evaluated whether testosterone undecanoate (TU), alone or combined with low dose cyproterone acetate (CPA), can maintain spermatogenic suppression induced by higher doses of CPA plus TU. Twenty-four men received for 12 wk 20 mg/d CPA plus 1000 mg/6 wk TU and then 1000 mg/8 wk TU plus 20 mg/d CPA (n = 8), 2 mg/d CPA (n = 8), or plus placebo (n = 8) for 32 wk. Blood samples, physical examinations, hormones, chemistry, hematology, semen analysis, and sexual/behavioral assessments were performed throughout the study. Sperm counts decreased to less than 1 million/ml in all subjects by wk 12, and 54% of them achieved azoospermia. Suppression of sperm counts was maintained until wk 44. Serum LH and FSH levels were suppressed by wk 12 of hormone administration and remained suppressed until wk 44. No significant changes in any biochemical parameters were detected at wk 44 in any group. There was a slight increase in total prostate volume to within the normal range at wk 44 that returned to baseline 1 yr after stopping hormone administration. In conclusion, TU alone or combined with lower doses of CPA maintains sperm suppression induced by higher dose CPA plus TU for 32 wk. This prototype regimen represents a promising male contraceptive regimen.

Phytoplasma: ecology and genomic diversity.

ABSTRACT The recent development of molecular-based probes such as mono- and polyclonal antibodies, cloned phytoplasma DNA fragments, and phytoplasma-specific primers for polymerase chain reaction (PCR) has allowed for advances in detection and identification of uncultured phytoplasmas (formerly called mycoplasma-like organisms). Comprehensive phylogenetic studies based on analysis of 16S ribosomal RNA (rRNA) or both 16S rRNA and ribosomal protein gene operon sequences established the phylogenetic position of phytoplasmas as members of the class Mollicutes, and the revealed phylogenetic interrelationships among phytoplasmas formed a basis for their classification. Based on restriction fragment length polymorphism (RFLP) analysis of PCR-amplified 16S rRNA gene sequences, phytoplasmas are currently classified into 14 groups and 38 subgroups that are consistent with groups delineated based on phylogenetic analysis using parsimony of 16S rRNA gene sequences. In the past decades, numerous phyto-plasma strains associated with plants and insect vectors have been identified using molecular-based tools. Genomic diversity of phytoplasma groups appears to be correlated with their sharing common insect vectors, host plants, or both in nature. The level of exchange of genetic information among phytoplasma strains in a given group is determined by three-way, vector-phytoplasma-plant interactions. A putative mechanism for the creation of new ecological niches and the evolution of new ecospecies is proposed.

Molecular identification of a new phytoplasma associated with alfalfa witches'-broom in oman.

ABSTRACT Alfalfa (Medicago sativa) plants showing witches'-broom symptoms typical of phytoplasmas were observed from Al-Batinah, Al-Sharqiya, Al-Bureimi, and interior regions of the Sultanate of Oman. Phytoplasmas were detected from all symptomatic samples by the specific amplification of their 16S-23S rRNA gene. Polymerase chain reaction (PCR), utilizing phytoplasma-specific universal primer pairs, consistently amplified a product of expected lengths when DNA extract from symptomatic samples was used as template. Asymptomatic plant samples and the negative control yielded no amplification. Restriction fragment length polymorphism profiles of PCR-amplified 16S-23S rDNA of alfalfa using the P1/P7 primer pair identified phytoplasmas belonging to peanut witches'-broom group (16SrII or faba bean phyllody). Restriction enzyme profiles showed that the phytoplasmas detected in all 300 samples belonged to the same ribosomal group. Extensive comparative analyses on P1/P7 amplimers of 20 phytoplasmas with Tru9I, Tsp509I, HpaII, TaqI, and RsaI clearly indicated that this phytoplasma is different from all the other phytoplasmas employed belonging to subgroup 16SrII, except tomato big bud phytoplasma from Australia, and could be therefore classified in subgroup 16SrII-D. The alfalfa witches'-broom (AlfWB) phytoplasma P1/P7 PCR product was sequenced directly after cloning and yielded a 1,690-bp product. The homology search showed 99% similarity (1,667 of 1,690 base identity) with papaya yellow crinkle (PapayaYC) phytoplasma from New Zealand. A phylogenetic tree based on 16S plus spacer regions sequences of 35 phytoplasmas, mainly from the Southern Hemisphere, showed that AlfWB is a new phytoplasma species, with closest relationships to PapayaYC phytoplasmas from New Zealand and Chinese pigeon pea witches'-broom phytoplasmas from Taiwan but distinguishable from them considering the different associated plant hosts and the extreme geographical isolation.