Microbial examination of anaerobic sludge adaptation to animal slurry.

The objective of this study was to evaluate changes in the microbial population of anaerobic sludge digesters during the adaptation to pig slurry (PS) using quantitative real-time polymerase chain reaction (qPCR) and qualitative scanning electron microscopy (SEM). Additionally, the relationship between microbial parameters and sludge physicochemical composition and methane yield was examined. Results showed that the addition of PS to an unadapted thermophilic anaerobic digester caused an increase in volatile fatty acids (VFA) concentration, a decrease in removal efficiency and CH4 yield. Additionally, increases in total bacteria and total archaea were observed using qPCR. Scanning electron micrographs provided a general overview of the sludge's cell morphology, morphological diversity and degree of organic matter degradation. A change in microbial morphotypes from homogeneous cell morphologies to a higher morphological diversity, similar to that observed in PS, was observed with the addition of PS by SEM. Therefore, the combination of qPCR and SEM allowed expanding the knowledge about the microbial adaptation to animal slurry in thermophilic anaerobic digesters.

Calculation of diagnostic parameters of advanced serological and molecular tissue-print methods for detection of Citrus tristeza virus: a model for other plant pathogens.

Citrus tristeza virus (CTV) is one of the most important virus diseases that affect citrus. Control of CTV is achieved by grafting selected virus-free citrus scions onto CTV-tolerant or -resistant rootstocks. Quarantine and certification programs are essential for avoiding the entry and propagation of severe strains of CTV. Citrus nurseries in Spain and central California (United States) maintain zero-tolerance policies for CTV that require sensitive, specific, and reliable pathogen-detection methods. Tissue-print (TP) real-time reverse-transcriptase polymerase chain reaction (RT-PCR) assay was compared with the validated TP enzyme-linked immunosorbent assay (ELISA), using the CTV-specific monoclonal antibodies 3DF1 and 3CA5, for CTV detection. In total, 1,395 samples from healthy and CTV-infected nursery and mature tree plants were analyzed with both methods. The total agreement between both detection methods was substantial (Cohen's kappa index of 0.77 ± 0.03). The diagnostic parameters of each technique (i.e., the sensitivity, specificity, and likelihood ratios) were evaluated in a second test involving 658 Citrus macrophylla nursery plants. Mexican lime indexing was used to evaluate samples with discrepant results in the analysis. For TP-ELISA, a sensitivity of 0.8015, a specificity of 0.9963, and a positive and negative likelihood ratio of 216.42 and 0.199, respectively, were estimated. For TP real-time RT-PCR, a sensitivity of 0.9820, a specificity of 0.8519, and a positive and negative likelihood ratio of 6.63 and 0.021, respectively, were estimated. These diagnostic parameters show that TP real-time RT-PCR was the most sensitive technique, whereas TP-ELISA showed the highest specificity, validating the use of the molecular technique for routine CTV-detection purposes. In addition, our results show that the combination of both techniques can accurately substitute for the conventional biological Mexican lime index for the detection of CTV. The calculation of diagnostic parameters is discussed, as a necessary tool, to validate detection or diagnostic methods in plant pathology. Furthermore, assessment of the post-test probability of disease after a diagnostic result and CTV prevalence allows selection of the best method for accurate and reliable diagnosis.

First Report of Arabis mosaic virus on Grapevine in Spain.

Arabis mosaic virus (ArMV; genus Nepovirus, family Comoviridae) is one of several nepoviruses responsible for infectious degeneration disease of grapevines in Europe (3). The first occurrence in Spain, in the summer of 2007, was found in Val de Salnés, Rias Baixas appellation, Galice on 25-year-old vines of the Albariño variety grafted onto an unidentified rootstock and showing leaf yellowing. The second finding was in the spring of 2008 in Barriobusto, Rioja appellation, Basque Country on 30-year-old vines of Tempranillo variety grafted onto 41B rootstock. In this case, no obvious foliar symptoms were observed but fruit set was very poor. Positive ELISA results were obtained at two different laboratories using antibodies to ArMV obtained from two companies (BIOREBA, Reinach, Switzerland and Sediag, Longvic, France). At a third lab, the presence of ArMV was further confirmed by reverse transcription (RT)-nested PCR using the primers described by Bertolini et al. (1). External primers ArMV 1 and ArMV 2 amplified a fragment of 340 bp from the coat protein region of the virus and internal primers ArMV i1 and ArMV i2 amplified a fragment of 203 bp. The specificity of the amplicons was subsequently confirmed by sequencing and comparison with other ArMV isolates available in the GenBank, EMBL, and DDBJ databases. Alignment performed using Blastn showed 85% nucleotide sequence identity with ArMV isolate NW (Accession No. AY017339). ELISA revealed co-infection with GLRaV-1 in Galice, GLRaV-3 in Rioja, and GFkV at both sites; these other viruses being common in their respective appellations. ArMV could be mechanically transmitted from rooted cuttings onto Chenopodium amaranticolor with an average of a 46% success rate (1:10 tissue/buffer ratio; [2]), but the range was very wide (0 to 100%) and dependent on the individual source vine. No statistical differences were found between nicotine or phosphate buffer for extraction or when using shoot tips or root tips as a source of virus (Fisher's exact test). Infection in C. amaranticolor was symptomless, but detectable by ELISA, and systemic. The Galician grapevine was an isolated plant, replanted on the spot of a dead one. Xiphinema diversicaudatum, the nematode vector of ArMV, was found in the vineyard soil. Only two ArMV-positive vines were found among 1,993 plants analyzed in Galice from 2005 to 2007 (no field data available for the second finding). In Rioja, one positive vine was found in a random sample of 74 vines from two different vineyards. Further testing of the neighboring vines indicated that one of the adjacent plants was also infected. This minimal spread since the vineyard was planted is suggestive of a lack of vectored transmission. In Spain as a whole, the virus seems to be rare and associated with the Atlantic biogeographic region. Both vineyards were planted before certified material became widely available. Currently, statutory testing of grapevine propagation material should prevent further spread. References: (1) E. Bertolini et al. Phytopathology 93:286, 2003. (2) G. P. Martelli, ed. Graft-Transmissible Diseases of Grapevines. Handbook for Detection and Diagnosis, FAO, Rome, 1993. (3) G. P. Martelli and E. Boudon-Padieu. Directory of Infectious Diseases of Grapevines and Viruses and Virus-like Diseases of the Grapevine. Bibliographic Report 1998-2004, CIHEAM, Paris, 2006.

Spinal cord compression complicating aseptic spondylodiscitis in ankylosing spondylitis.

Aseptic spondylodiscitis is a well recognized complication of ankylosing spondylitis. Neurological complications of such discovertebral lesions are uncommon. We report a new case with a diagnosis of T12-L1 spondylodiscitis which developed ten years after a spinal cord compression. Such neurological complications of aseptic spondylodiscitis may be explained by proliferative epidural tissue without predominant inflammatory infiltrates and also the development of new bone reaction, suggesting the contribution of mechanical factors.

Graft transmission efficiencies and multiplication of 'Candidatus Liberibacter americanus' and 'ca. Liberibacter asiaticus' in citrus plants.

In Brazil 'Candidatus Liberibacter asiaticus' and 'Ca. L. americanus' cause huanglongbing (also known as greening), the most destructive citrus disease. A shift in pathogen prevalence was observed over time, with a disproportional increase in 'Ca. L. asiaticus' occurrence. Graft transmission experiments were used for a comparative study of both species using budsticks from symptomatic branches of field-affected trees as inoculum. The plants were inoculated with 'Ca. L. asiaticus' or 'Ca. L. americanus' alone, or simultaneously with both species. Symptom manifestation and conventional and quantitative real-time polymerase chain reaction were used for plant evaluations. 'Ca. L. americanus' was detected mainly in symptomatic plants and 'Ca. L. asiaticus' was detected in symptomatic plants as well as in infected plants prior to symptom manifestation. Transmission percentages varied from 54.7 to 88.0% for 'Ca. L. asiaticus' and 10.0 to 45.2% for 'Ca. L. americanus' in two experiments. In co-inoculated plants, 12.9% contained 'Ca. L. americanus' only, 40.3% contained 'Ca. L. asiaticus' only, and 19.3% contained both species. Average bacterial titers for 'Ca. L. asiaticus' and 'Ca. L. americanus', in log cells per gram of leaf midrib, were 6.42 and 4.87 for the experimental plants and 6.67 and 5.74 for the field trees used as the source of inoculum. The higher bacterial populations of the 'Ca. L. asiaticus'-infected plants provided an explanation for the disproportional increase in field prevalence of this species over time, based on the greater likelihood for pathogen transmission by the insect vector.

Liberibacters Associated with Citrus Huanglongbing in Brazil: 'Candidatus Liberibacter asiaticus' Is Heat Tolerant, 'Ca. L. americanus' Is Heat Sensitive.

In São Paulo State, Brazil, 'Candidatus Liberibacter americanus' and 'Candidatus Liberibacter asiaticus' are associated with huanglongbing (HLB). Affected municipalities occur mainly in the central and southern regions, where the annual number of hours above 30°C is two to five times lower than that in the extreme northern and western regions. The influence of temperature on sweet orange trees infected with 'Ca. L. asiaticus' or 'Ca. L. americanus' was studied in temperature-controlled growth chambers. Symptom progression on new shoots of naturally infected and experimentally graft-inoculated symptomatic sweet orange trees was assessed. Mottled leaves developed on all infected trees at 22 to 24°C, but not on any 'Ca. L. americanus'-infected trees at 27 to 32°C. Quantitative, real time-PCR was used to determine the liberibacter titers in the trees. After 90 days, 'Ca. L. asiaticus'-infected trees had high titers at 32 and 35°C, but not at 38°C, while 'Ca. L. americanus'-infected trees had high titers at 24°C, but at 32°C the titers were very low or the liberibacters could not be detected. Thus, the multiplication of 'Ca. L. asiaticus' is not yet affected at 35°C, while a temperature of 32°C is detrimental to 'Ca. L. americanus'. Thus, 'Ca. L. americanus' is less heat tolerant than 'Ca. L. asiaticus'. The uneven distribution of these two liberibacters in São Paulo State might be in relation with these results.

Integrated approach for detection of nonculturable cells of Ralstonia solanacearum in asymptomatic Pelargonium spp. cuttings.

Ralstonia solanacearum (biovar 2, race 3) is a soil and water-borne pathogen that causes serious diseases in several solanaceous hosts. It can also infect geranium plants, posing an important threat to their culture when latently infected cuttings are imported from countries where the pathogen is endemic. R. solanacearum can be present in very low numbers in asymptomatic geranium cuttings, and/or in a particular stressed physiological state that escapes direct isolation on the solid media usually employed. Consequently, an integrated protocol has been developed to analyze asymptomatic geranium cuttings routinely. The first screening tests include isolation and co-operational-polymerase chain reaction (Co-PCR), based on the simultaneous and co-operational action of three primers from 16S rRNA of R. solanacearum. This method was selected as the most sensitive one, able to detect only 1 cell/ml including nonculturable cells. When isolation is negative but Co-PCR is positive, the bioassay in tomato plants is proposed, since stressed bacterial cells or those present in low numbers that do not grow on solid media can be recovered from inoculated tomato plants and retain pathogenicity. This methodology has been demonstrated to be useful and has allowed us to assess the relevance of the physiological status of bacterial cells and its implications in detection. It also reveals the risk of introducing R. solanacearum through asymptomatic geranium material when relying only on bacterial isolation.

Factors Affecting Pseudomonas savastanoi pv. savastanoi Plant Inoculations and Their Use for Evaluation of Olive Cultivar Susceptibility.

ABSTRACT Pseudomonas savastanoi pv. savastanoi causes olive knot disease, which is present in most countries where olive trees are grown. Although the use of cultivars with low susceptibility may be one of the most appropriate methods of disease control, little information is available from inoculation assays, and cultivar susceptibility assessments have been limited to few cultivars. We have evaluated the effects of pathogen virulence, plant age, the dose/response relationship, and the induction of secondary tumors in olive inoculation assays. Most P. savastanoi pv. savastanoi strains evaluated were highly virulent to olive plants, but interactions between cultivars and strains were found. The severity of the disease in a given cultivar was strongly dependent of the pathogen dose applied at the wound sites. Secondary tumors developed in noninoculated wounds following inoculation at another position on the stem, suggesting the migration of the pathogen within olive plants. Proportion and weight of primary knots and the presence of secondary knots were evaluated in 29 olive cultivars inoculated with two pathogen strains at two inoculum doses, allowing us to rate most of the cultivars as having either high, medium, or low susceptibility to olive knot disease. None of the cultivars were immune to the disease.

Antibodies to hepatitis C virus in primary biliary cirrhosis.

We investigated the prevalence of anti-HCV in 160 consecutive patients with primary biliary cirrhosis. By ELISA, 19 (12%) were positive, as compared to a 68% prevalence in 135 patients with chronic non-A, non-B hepatitis. Serum IgG levels were significantly higher in the anti-HCV positive group. By RIBA, seropositivity was confirmed for 4 patients, whereas 7 were indeterminate. A slight, non-significant reduction of life expectancy was found in anti-HCV positive patients. Until reliable and independent confirmatory tests become available, definitive conclusions on the importance of anti-HCV positivity in primary biliary cirrhosis are improper.

Influence of TRH on regional blood flow and metabolic acidosis in a model of volume-controlled hemorrhagic shock in rats.

In anesthetized rats, massive bleeding to a severe condition of hemorrhagic shock (invariably leading to death within 30 min) was obviously associated with a dramatic decrease in tissue blood flow and with profound modifications of several blood parameters leading to metabolic acidosis: decrease in arterial and venous pH, bicarbonate and BE, decrease in arterial pCO2 and in venous pO2 and SO2, increase in arterial pO2, venous pCO2 and venous lactate. The i.v. bolus injection of protirelin tartrate (TRH-T, 4 mg/kg), which produces a prompt and sustained reversal of the shock condition, caused a rapid increase in venous pO2, pCO2 and SO2; on the other hand, arterial and venous pH, bicarbonate and BE continued to decrease--and venous lactate to increase during the first few minutes after treatment. However venous pCO2 and lactate, as well as arterial and venous pH, returned to the pre-bleeding values within 60 min after treatment. The data are in keeping with the TRH-T-induced improvement of circulatory and respiratory functions, with mobilization of the residual blood from its capillary pooling and consequent immission of acid metabolites into the blood stream.

Single-step multiplex RT-PCR for simultaneous and colourimetric detection of six RNA viruses in olive trees.

A single-step multiplex RT-PCR was developed for the simultaneous and colourimetric detection of six RNA viruses (Cucumber mosaic virus, Cherry leaf roll virus, strawberry latent ringspot virus, Arabis mosaic virus, Olive latent-1 virus and Olive latent-2 virus) which infect olive trees. Six compatible primer set for one-step RT-PCR amplification in a single closed-tube and 3' digoxigenin labelled probes were designed in optimal, specific and conserved regions. The method has been assessed with 195 Spanish field olive trees, suggesting that approximately 1.5% of the tested material was infected by Cucumber mosaic virus and 0.5% by Cherry leaf roll virus. This method saves time and reagent costs compared with monospecific RT-PCR which needs several reactions for the same number of tests. Using colourimetric detection, it is possible to analyse many samples, it increases sensitivity 10-fold, and whilst facilitating the interpretation of results, it avoids the use of gels and the toxic ethidium bromide. The method could be used routinely for sanitary and certification programmes.

TRH reverses the ECG and EEG ischemic changes induced by massive hemorrhage in rats.

In a rat model of volume-controlled hemorrhagic shock causing the death of all saline-treated animals within 30 min of treatment, the intravenous bolus injection of thyrotropin- releasing hormone tartrate (TRH-T) at the dose of 4 mg/kg induced the prompt and sustained disappearance of the ECG and EEG signs of heart and brain ischemia, along with the reversal of hypotension and respiratory depression and with 100% survival rate at the end of the 2 h observation period. These data confirm that, in a pre-terminal condition induced by massive hemorrhage, timely treatment with TRH-T will restore heart and brain perfusion to levels compatible with survival and with functional recovery from ischemia and maintain it at those levels for some hours.

Influence of ACTH-(1-24) on metabolic acidosis and hypoxemia induced by massive hemorrhage in rats.

In anesthetized rats, step-wise bleeding to a severe condition of hemorrhagic shock causes a decrease in arterial and venous pH and in venous PO2 and SO2 and an increase in arterial PO2 and in venous PCO2 and lactic acid. The intravenous bolus injection of ACTH-(1-24) (160 micrograms/kg)--which causes a rapid and sustained reversal of the shock condition--produces a gradual and almost complete recovery (within 60 min) of venous PO2, PCO2 and SO2; on the other hand, the normalization of blood pH and lactate is preceded by a further worsening during the first minutes after treatment. On the whole, these data are compatible with the ACTH-(1-24)-induced mobilization of the residual blood--which is pooled in poorly oxygenated tissues--and with the improved circulatory and respiratory functions.

Safety and efficacy of laparoscopic radiofrequency ablation of hepatocellular carcinoma in patients with liver cirrhosis.

BACKGROUND: The optimal treatment for hepatocellular carcinoma (HCC) is surgical resection. However, only a small percentage of patients are operative candidates. Percutaneous radiofrequency interstitial thermal ablation (RITA) has been shown to be efficacious in the treatment of unresectable HCC. Recent advances in laparoscopic ultrasound have greatly improved the accuracy in detecting intrahepatic HCC nodules, many of which were missed by computed tomography. Our objective was to introduce a novel operative combination of laparoscopic ultrasound with laparoscopic RITA in the treatment of HCC. METHODS: Eighty-eight patients with HCC in liver cirrhosis were submitted to laparoscopic RITA under sonographic guide. Most patients were in Child's A class of liver function. Patients with large tumors (> 5 cm), portal vein thrombosis, or severe liver disease (Child's C class) were excluded. RESULTS: The laparoscopic RITA procedure was completed in 86 of 88 patients (98% feasibility rate). Laparoscopy with laparoscopic ultrasound identified 23 new malignant lesions (27%) in comparison with the results of preoperative imaging. A total of 127 lesions were treated by RITA. There was no operative mortality. Sixty-one patients had no complication (71%). After a mean follow-up of 14.3 +/- 11.6 months, a complete response with a 100% necrosis was achieved in 70 of 83 patients examined (86%). During follow-up, 9 patients (11%) locally recurred at the RITA site and 38 patients (46%) had new malignant nodules. CONCLUSIONS: Laparoscopic RITA of HCC proved to be a safe and effective technique in the short term. This technique may be indicated when the percutaneous approach to the lesion is very difficult or if the patient is too ill to undergo laparotomy.

Laparoscopic radiofrequency of hepatocellular carcinoma (HCC) in liver cirrhosis.

BACKGROUND/AIMS: In this report, the feasibility and efficacy of laparoscopic radiofrequency interstitial thermal ablation of hepatocellular carcinoma has been evaluated in 20 patients. METHODOLOGY: The laparoscopic approach with the use of intraoperative ultrasonography allowed us to obtain additional information regarding liver nodules and a complete treatment of the liver lesions. RESULTS: The complication rate was low and there was no operative mortality. A complete necrosis has been obtained in 90% of the cases at 1 month dynamic computed tomography following the treatment. CONCLUSIONS: Laparoscopic radiofrequency thermal ablation of hepatocellular carcinoma proved to be a safe and effective technique; its use may be proposed in selected patients. Larger series are needed to accurately assess its role among the other ablative therapies of hepatocellular carcinoma.

[Endogenous anti-analgesic systems]. / Sistemi anti-analgesici endogeni.

Nociception is of vital importance for the organism, while its inhibition by endogenous opioid systems is usually a sign of surrender. Therefore, it must be assumed that endogenous analgesic systems are balanced, and in fact, under normal conditions, overwhelmed, by teleologically far more important anti-analgesic systems. The two main anti-analgesic systems--i.e., the melanotropinergic and the cholecystokininergic--are here reviewed for their role, not only in nociception, but in a wide variety of vital functions (endocrine, gastrointestinal, ingestive, reproductive, cardiovascular, immune, etc.). Available data strongly suggest that these systems (particularly the melanotropinergic one) play a key role in the overall homeostasis of the body. Moreover, modulation of endogenous anti-analgesic systems may disclose a new, unforeseen approach to the treatment of pain.

[The treatment of severe, therapy-resistant depression using infusions of antidepressants]. / Die Behandlung schwerer therapieresistenter Depressionen mit Infusion von Antidepressiva.

Infusion-therapy with antidepressants has been of value in severe as well as therapy resistant depressive states. In addition to doses lower than those used for oral treatment, a more rapid onset of therapeutic effect and a better tolerance, the beneficial effect seems also to depend on the setting in which the treatment takes place. Infusion-therapy is a combined pharmacopsychotherapeutic procedure. Next to infusion-treatment a pretreatment with neuroleptics is advised either via the oral or parenteral route. In extremely refractory depression the infusion-therapy can be applied twice a day; in some cases we resort to continuous infusion for a few days. Infusion-therapy is not applicable in patients prone to epileptic seizures or with serious cerebral dysfunction with a risk of delirium. In case of doubt an EEG is mandatory. Tricyclic antidepressants may not be used in cardiac diseases especially those with troubles of the conduction propagation or repolarization.

Intraoperative ultrasound patterns predict recurrences after surgical treatments for hepatocellular carcinoma().

INTRODUCTION: Hepatocellular carcinoma (HCC) is associated with a high incidence of postoperative recurrence, despite high rates of complete necrosis with radiofrequency ablation (RFA) and curative hepatic resections (HR). The aim of this study was to identify intraoperative ultrasound patterns observed during HR or RFA that predicting intrahepatic HCC recurrence. MATERIALS AND METHODS: From January 1997 through August 2008, we treated 377 patients with HCC (158 with HR and 219 with surgical RFA). All patients underwent intraoperative ultrasound (IOUS) examination. Primary HCCs was classified according to diameter, HCC pattern (nodular or infiltrative), echogenicity (hyper- or hypo-), echotexture (homogeneous or inhomogeneous), capsular invasion, mosaic pattern, nodule-in-nodule appearance, and infiltration of portal vessels. Number of HCC nodules was also considered. Comparisons between the groups of possible factors for intrahepatic recurrence of treated tumors were performed using the Kaplan-Meier method and compared using the log-rank test. RESULTS: Patients were followed for 9-127 months (median: 18.6 months), and intrahepatic recurrence was observed in 198 (52.5%). In 138 patients (36.5%), recurrences were located in different segments with respect to the primary tumor. In 60 HCC tumors (16%), local recurrences were found in the same segment as the primary tumor. At univariate analysis, primary HCC echogenicity and mosaic pattern were the only factors not significant associated with intrahepatic recurrences. CONCLUSION: IOUS is an accurate staging tool for use during "surgical" resection or RFA. This study shows that IOUS patterns can also be used to estimate the risk of post-treatment HCC recurrence. In patients at high risk for this outcome, closer follow-up and use of adjuvant therapies could be useful.

Failure of ursodeoxycholic acid to prevent a cholestatic episode in a patient with benign recurrent intrahepatic cholestasis: a study of bile acid metabolism.

Ursodeoxycholic acid was administered to a patient with benign recurrent intrahepatic cholestasis to prevent cholestatic episodes. A detailed study of bile acid metabolism in this patient was carried out in the anicteric and icteric phases before and after ursodeoxycholic acid (750 mg/day) administration. Urinary, biliary and serum bile acids were measured by gas chromatography-mass spectrometry and by high-performance liquid chromatography techniques. During the anicteric phase the daily urinary excretion and serum concentrations of bile acids were within normal ranges, indicating normal hepatic uptake and secretion of bile acids during the cholestasis-free period. Only slight qualitative differences from normal individuals were observed; the relative proportions of deoxycholic acid in the bile and serum were higher, and 12-oxo-lithocholic acid was the predominant urinary bile acid. During the icteric phase a marked increase in the urinary excretion of primary bile acids and C-1, C-2, C-4 and C-6 hydroxylated metabolites was found. Serum bile acid concentrations increased before the rise in bilirubin, suggesting an acute disturbance in bile acid transport at the onset of the cholestatic attack. After ursodeoxycholic acid administration in the anicteric phase, bile became enriched with the exogenous bile acid, but little qualitative change was found in the other metabolites present in the urine, serum or bile during the anicteric or icteric phases. Prolonged administration of ursodeoxycholic acid failed to prevent recurrence of a cholestatic episode, suggesting that in benign recurrent intrahepatic cholestasis, oral ursodeoxycholic acid may be of little benefit in the treatment or prevention of cholestasis despite marked enrichment of the bile acid pool with this hydrophilic bile acid.

Detection of Pseudomonas savastanoi pv. savastanoi in olive plants by enrichment and PCR.

The sequence of the gene iaaL of Pseudomonas savastanoi EW2009 was used to design primers for PCR amplification. The iaaL-derived primers directed the amplification of a 454-bp fragment from genomic DNA isolated from 70 strains of P. savastanoi, whereas genomic DNA from 93 non-P. savastanoi isolates did not yield this amplified product. A previous bacterial enrichment in the semiselective liquid medium PVF-1 improved the PCR sensitivity level, allowing detection of 10 to 100 CFU/ml of plant extract. P. savastanoi was detected by the developed enrichment-PCR method in knots from different varieties of inoculated and naturally infected olive trees. Moreover, P. savastanoi was detected in symptomless stem tissues from naturally infected olive plants. This enrichment-PCR method is more sensitive and less cumbersome than the conventional isolation methods for detection of P. savastanoi.