Diversity, distribution and multi-functional attributes of bacterial communities associated with the rhizosphere and endosphere of timothy (Phleum pratense L.).

AIMS: To characterize the bacterial communities of the rhizosphere and endosphere of the forage grass timothy (Phleum pratense L.) and evaluate the functional attributes with respect to growth promotion properties, antimicrobial and biosurfactant capacities. METHODS AND RESULTS: A total of 254 culturable bacteria were identified using 16S rRNA sequencing and grouped into 16 taxa that shared high homology of 98-99% with other known sequences. A majority of the isolates were recovered from the rhizosphere soil fraction and leaf and crown tissues. Bacillus genus was the most abundant in the bulk and rhizosphere soil fractions. Isolates belonging to the Methylobacterium genus were exclusively found in leaves making them tissue-specific. A majority of the bacterial isolates exhibited multi-functional growth promotion attributes and plant stress improvement related to the production of indole 3-acetic acid, VOC and siderophores and polymer-degrading enzymes and 1-aminocyclopropane-1-carboxylate (ACC) deaminase activities. Some demonstrated antimicrobial properties such as hydrogen cyanide and biosurfactant production and activities of fungal cell wall degrading enzymes. The internalization and spread of selected bacterial isolates in timothy seedlings under gnotobiotic conditions was confirmed using the culture-dependent method and SEM microscopy in proof-of-concept experiments. CONCLUSIONS: The attributes of some isolates with respect to growth promotion abilities, biocontrol potential and efficient colonization of timothy make them desirable for future development as potential biofertilizer tools. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides the first evidence of bacterial endophytes that have the necessary functional attributes to protect cool-season forage grasses against abiotic stress.

Fusarium graminearum1H NMR metabolomics.

Raw 1H NMR spectra of Fusarium graminearum hyphae can be found at the website of the pesticide metabolomics group (PMG) of the Agricultural University of Athens at the address: http://www.aua.gr/pesticide-metabolomicsgroup/Resources/Fusarium_graminearum_NMR_spectra.html, accession number PMG-01-17. The data set support the research article "Implication of Fusarium graminearum Primary Metabolism in its Resistance to Benzimidazole Fungicides as revealed by 1H NMR Metabolomics" [1].

Implication of Fusarium graminearum primary metabolism in its resistance to benzimidazole fungicides as revealed by 1H NMR metabolomics.

Fungal metabolomics is a field of high potential but yet largely unexploited. Focusing on plant-pathogenic fungi, no metabolomics studies exist on their resistance to fungicides, which represents a major issue that the agrochemical and agricultural sectors are facing. Fungal infections cause quantitative, but also qualitative yield losses, especially in the case of mycotoxin-producing species. The aim of the study was to correlate metabolic changes in Fusarium graminearum strains' metabolomes with their carbendazim-resistant level and discover corresponding metabolites-biomarkers, with primary focus on its primary metabolism. For this purpose, comparative 1H NMR metabolomics was applied to a wild-type and four carbendazim-resistant Fusarium graminearum strains following or not exposure to the fungicide. Results showed an excellent discrimination between the strains based on their carbendazim-resistance following exposure to low concentration of the fungicide (2 mg L-1). Both genotype and fungicide treatments had a major impact on fungal metabolism. Among the signatory metabolites, a positive correlation was discovered between the content of F. graminearum strains in amino acids of the aromatic and pyruvate families, l-glutamate, l-proline, l-serine, pyroglutamate, and succinate and their carbendazim-resistance level. In contrary, their content in l-glutamine and l-threonine, had a negative correlation. Many of these metabolites play important roles in fungal physiology and responses to stresses. This work represents a proof-of-concept of the applicability of 1H NMR metabolomics for high-throughput screening of fungal mutations leading to fungicide resistance, and the study of its biochemical basis, focusing on the involvement of primary metabolism. Results could be further exploited in programs of resistance monitoring, genetic engineering, and crop protection for combating fungal resistance to fungicides.

Isolation and characterization of indigenous endophytic bacteria associated with leaves of switchgrass (Panicum virgatum L.) cultivars.

AIMS: To isolate and characterize indigenous bacterial endophytes from cultivars of switchgrass and study their antimicrobial and growth promoting potential. METHODS AND RESULTS: The diversity, molecular and biochemical characterizations of indigenous and culturable bacterial endophytes residing in leaves of switchgrass have not been studied previously. This study describes the characterization of 31 bacterial endophytes from three switchgrass cutlivars: Cave-in Rock, Blue Jacket and Tecumseh. Molecular and phylogenetic analysis based on the 16S rRNA sequence grouped the endophytes into eight different taxa that shared high homology of 98-99% with other known sequences. Bacterial endophytes were identified as Microbacterium testaceum, Curtobacterium flaccumfaciens, Bacillus subtilis and Bacillus pumilus, Pseudomonas fluorescens, Sphingomonas parapaucimobilis, Serratia sp. and Pantoea ananatis. Some endophytes were detected in switchgrass seeds and in plants that originated from seeds collected a year earlier, confirming vertical transmission to the next generation of the host. Selected endophytes produced cellulases and were capable of solubilizing inorganic phosphorus. Analysis of cell-free culture filtrate of selected strains using direct infusion orbitrap mass spectrometry confirmed the presence of several well-characterized lipopeptide toxins and phytohormones. Re-inoculation of the roots of switchgrass seedlings with endophytes singly or combined confirmed their migration to the upper aerial parts of the plant. CONCLUSIONS: Our findings suggest that switchgrass leaves harbour a diversity of bacterial endophytes, some of which could potentially be applied as growth promoting bacteria. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report on the characterization of indigenous bacterial endophytes and their potential use as biofertilizers.

Should central serous chorioretinopathy be added to the list of ocular side effects of phosphodiesterase 5 inhibitors?

INTRODUCTION: Rare cases of central serous chorioretinopathy (CSC) associated with the intake of sildenafil citrate have been reported, although CSC is not included in the list of phosphodiesterase 5 (PDE5) inhibitor side effects. MATERIALS AND METHODS: We present a review of the literature and 2 cases of CSC in 2 men taking PDE5 inhibitors (vardenafil and tadalafil) for erectile dysfunction. In both cases chorioretinopathy appeared after intake of the inhibitor, resolved once the latter was discontinued, reappeared when the inhibitor was restarted and resolved once again after the inhibitor had been discontinued for the second time. DISCUSSION: PDE5 inhibitors used for male erectile dysfunction have been associated with ocular side effects including lid edema, hyposphagma, photophobia, mydriasis, dyschromatopsia, and nonarteritic anterior ischemic optic neuropathy. CSC was previously described in patients taking sildenafil citrate. Very recently, a case of CSC after tadalafil intake was reported. The relevant literature is reviewed and possible pathophysiologic mechanisms are discussed. CONCLUSION: The 2 presented cases of CSC after intake of vardenafil or tadalafil with positive dechallenge, rechallenge and second dechallenge reactions provide important arguments for considering CSC as a rare PDE5 inhibitor class-specific side effect.

Differentiating Alström from Bardet-Biedl syndrome (BBS) using systematic ciliopathy genes sequencing.

INTRODUCTION: Early onset retinal degeneration associated with obesity can present a diagnostic challenge in paediatric ophthalmology practice. Clinical overlap between Bardet-Biedl syndrome (BBS) and Alström syndrome has been described, although the two entities are genetically distinct. To date, 16 genes are known to be associated with BBS (BBS1-16) and only one gene has been identified for Alström syndrome (ALMS1). MATERIALS AND METHODS: In collaboration with the French National Center for Sequencing (CNS, Evry), all coding exons and flanking introns were sequenced for 27 ciliopathy genes (BBS1-12, MGC1203, TTC21b, AHI1, NPHP2-8 (NPHP6=BBS14), MKS1(BBS13), MKS3, C2ORF86, SDCCAG8, ALMS1) in 96 patients referred with a clinical diagnosis of BBS. ALMS1 gene analysis included sequencing of all coding exons. RESULTS: BBS known gene mutations were found in 44 patients (36 with two mutations and 8 heterozygous). ALMS1 mutations were found in four cases. The rate of ALMS1 mutations among patients suspected of having BBS was 4.2%. DISCUSSION: Clinically, all four patients presented early-onset severe retinal degeneration with congenital nystagmus associated with obesity. The difficult early differential diagnosis between the two syndromes is outlined. One mutation had already been reported (c.11310delAGAG/p.R3770fsX) and three were novel (c.2293C > T/p.Q765X, c.6823insA/p.R2275fsX, c.9046delA/p.N3016fsX). CONCLUSIONS: Ciliopathy genes sequencing can be very helpful in providing a timely diagnosis in this group of patients, hence appropriate genetic counselling for families and adequate medical follow-up for affected children.

[Multifocal electroretinography in follow-up of patients treated with hydroxychloroquine]. / L'apport de l'électrorétinographie multifocale dans le suivi des patients traités par hydroxychloroquine.

INTRODUCTION: Ophthalmologic follow-up of hydroxychloroquine-treated patients is of major importance because of the risk for potentially irreversible retinal toxicity. The use of the multifocal electroretinogram for the follow-up of such patients has already been reported. We studied the capacity of the multifocal electroretinogram to detect early signs of retinal toxicity. PATIENTS AND METHODS: This study consisted of multifocal electroretinogram analysis of 28 patients treated with hydroxychloroquine for a period of 6-72 months. All patients selected had a normal clinical and paraclinical ophthalmological evaluation. A multifocal electroretinogram according to the international recommendations was obtained. We analyzed the amplitude and the implicit time for kernel 1 responses by quadrant and by ring. RESULTS: For both eyes, in both ring and quadrant analyses, the average implicit times of patients receiving hydroxychloroquine were significantly elevated (p<0.01). On the other hand, the amplitudes were not significantly attenuated. In a total of 28 patients, seven (25%) presented a significant prolongation of the implicit time and significant amplitude attenuation was found in only one patient. DISCUSSION: Implicit time prolongation in hydroxychloroquine-treated patients has already been reported. However, dissociation between amplitude and implicit time changes has, to our knowledge, never been previously described. This observation is important given that numerous studies focus exclusively on amplitude. CONCLUSION: The multifocal electroretinogram of patients receiving hydroxychloroquine can present lengthened kernel 1 implicit time, despite otherwise normal ophthalmological evaluation. Further studies are necessary to evaluate the prognostic value of mfERG responses in the early detection of patients presenting a retinotoxicity risk.

A novel nonsense B3GALTL mutation confirms Peters plus syndrome in a patient with multiple malformations and Peters anomaly.

Peters plus syndrome is an autosomal recessive rare congenital disorder defined by corneal Peters anomaly with short disproportionate stature, development delay and dysmorphic facial features. In addition, cardiac, genito-urinary and/or central nervous system malformations can be present. Mutations in the beta-1,3-galactosyltransferase-like glycosyltransferase gene (B3GALTL) have been reported in patients with Peters plus syndrome prompting phenotype-genotype studies because of the variable clinical spectrum related to the syndrome. A 20 month old boy presenting with bilateral Peters anomaly in association with multiple developmental anomalies including cerebral malformations was found to carry a novel homozygous B3GALTL nonsense mutation [p.Tyr366X]. This is the first stop mutation described in association with this gene. The present report confirms the wide clinical spectrum of Peters plus syndrome, underlines the major clinical criteria of the syndrome and the major implication of B3GALTL gene in this condition. Ophthalmologic examination in multiple developmental anomalies remains an important clinical issue that may lead to specific gene screening. In Peters plus syndrome B3GALTL molecular test provides diagnosis confirmation and improves dramatically genetic counselling for the families.

De novo duplication of MECP2 in a girl with mental retardation and no obvious dysmorphic features.

Loss-of-function mutations of MECP2 are responsible for Rett syndrome (RTT), an X-linked neurodevelopmental disorder affecting mainly girls. The availability of MECP2 testing has led to the identification of such mutations in girls with atypical RTT features and the recognition of milder forms. Furthermore, duplication of the entire gene has recently been described in boys with mental retardation and recurrent infections. We describe a girl with a heterozygous de novo MECP2 duplication. The patient, at the age of 19, has mental retardation with no autistic features. She is friendly but gets frequently anxious. She has neither dysmorphic features nor malformations. Her motor development was delayed with walking at 20 months. Speech is fluid with good pronunciation but is simple and repetitive. Diagnosis was made after single-strand conformation analysis (SSCA) and multiplex ligation-dependent probe amplification (MLPA) analysis of MECP2. Array comparative genomic hybridization (aCGH) analysis showed a duplication of 29 kb including MECP2 and part of IRAK1. Fluorescent in situ hybridization (FISH) has revealed that the duplicated region is inserted near the telomere of the short arm of chromosome 10. X-chromosome inactivation in leukocyte DNA was not skewed. We conclude that it is likely that this MECP2 duplication is responsible for the mental retardation in this patient. This case broadens the phenotypic spectrum of MECP2 abnormalities with consequent implication in diagnosis and genetic counselling of girls with non-syndromic mental retardation.

Comparative toxicity of the phytotoxins (8R,16R)-(-)-pyrenophorin and (5S,8R,13S,16R)-(-)-pyrenophorol on aquatic organisms.

The acute toxicities of the fungal phytotoxins (8R,16R)-(-)-pyrenophorin and (5S,8R,13S,16R)-(-)-pyrenophorol on Vibrio fischeri, Oscillatoria perornata, Pseudokirchneriella subcapitata, Lemna minor and Artemia fransiscana were evaluated. (8R,16R)-(-)-pyrenophorin was more toxic than (5S,8R,13S,16R)-(-)-pyrenophorol to V. fischeri, O. perornata, L. minor and A. fransiscana. The highest acute toxicity of (8R,16R)-(-)-pyrenophorin was exhibited on V. fischeri (5 min median effective concentration of 3.57 M 10(-5)) whereas the corresponding value for (5S,8R,13S,16R)-(-)-pyrenophorol was 801 M 10(-5). P. subcapitata exhibited a lack of sensitivity (median inhibitory concentration of >10 M 10(-5)) to both phytotoxins.

Physiological and ultrastructural changes in "green islands" on Avena sterilis leaves caused by (8R,16R)-(-)-pyrenophorin.

The biochemical and ultrastructural changes in "green islands" (GIs) on detached Avena sterilis leaves caused by the macrodiolide (8R,16R)-(-)-pyrenophorin in the dark were examined. In the absence of light, leaf segments retained their photosynthetic pigments for 96 h after treatment with (8R,16R)-(-)-pyrenophorin (70 muM), whereas in the untreated leaves complete senescence, loss of photosynthetic pigments and cell disorganization were observed 72 h after detachment. Proteolytic enzyme activity in treated tissues with pyrenophorin remained at low levels for 96 h after treatment and protein dissipation was lower in the treated than in the untreated. Although tissues in "GIs" seem macroscopically healthy, electron microscopy observations revealed structurally disorganized cells filled with granular, electron-dense material. Chloroplasts were severely damaged and contained a large number of plastoglobuli. Similar ultrastructural changes were also observed in A. sterilis tissues treated with the phytotoxin under illumination, indicating a mechanism operating both under illumination and in the dark.

Pattern of treatment among diabetic patients in a sample of urban Greek population.

It is well known that therapeutic habits and diabetic patient's compliance differ substantially between different populations. It is also accepted that if patients are recruited in specialized diabetes centers some important bias is likely to occur, e.g. an excess of insulin-treated subjects. Therefore, population studies are very helpful in order to establish treatment patterns in different populations. A descriptive study was performed during an epidemiological survey conducted on 12,842 randomly selected inhabitants of a suburb of Athens (both sexes, all ages). 509 persons were recorded as known diabetics. Information concerning the type of treatment was collected. Reliable data were available on 490 diabetic individuals. 145 (29.6%) of them were following diet alone, while 270 (55.1%) were receiving antidiabetic tablets and only 75 (15.1%) injected insulin. In patients 40 years old or more (n = 471), the corresponding numbers were 30.1, 56.5 and 13.4% respectively. Thus, it is obvious that insulin-treated patients represent a small percentage of the total diabetic population. It seems that these differences are caused by genetic as well as other factors.

Evidence for an increase in the prevalence of known diabetes in a sample of an urban population in Greece.

It is accepted that the prevalence of known diabetes mellitus has increased in some countries, although the available data are incomplete and sometimes anecdotal. In 1974 a sample of the urban population (n = 21,410) in a suburb of Athens (Greece) was evaluated for the prevalence of known diabetes mellitus. The study was repeated 17 years later in the same area under the same conditions and methodology on 12,836 randomly selected inhabitants (both sexes, all ages). Visiting nurses contacted the inhabitants at home and filled in a questionnaire. The results show a clear increase in diabetes prevalence of the population as a whole (standardized rates: 1974: 2.4% (95% confidence intervals 2.2-2.6%) 1990: 3.1 (2.8-3.4%)). Standardization was done on the basis of data obtained from the same census. The changes were statistically significant in the 50-59, 60-69, and 70-79 age groups (1974: 5.87, 10.98, and 11.41%; 1990: 7.70, 13.68, and 19.52%, respectively). Thus, the prevalence of known diabetes mellitus has considerably increased during the last 17 years in a sample of urban Greek population.