First Study Case of Microbial Biocontrol Agents Isolated from Aquaponics Through the Mining of High-Throughput Sequencing Data to Control Pythium aphanidermatum on Lettuce.

Aquaponics is defined as a sustainable and integrated system that combines fish aquaculture and hydroponic plant production in the same recirculated water loop. A recent study using high-throughput sequencing (HTS) technologies highlighted that microbial communities from an aquaponic system could control one of the most problematic pathogens in soilless lettuce culture, namely, Pythium aphanidermatum. Therefore, this study aims at isolating the microorganisms responsible for this biocontrol action. Based on the most promising genera identified by HTS, an innovative strategy for isolating and testing original biocontrol agents from aquaponic water was designed to control P. aphanidermatum. Eighty-two bacterial strains and 18 fungal strains were isolated, identified by Sanger sequencing, and screened in vivo to control damping-off of lettuce seeds caused by P. aphanidermatum. Out of these 100 isolates, the eight most efficacious ones were selected and further tested individually to control root rot disease caused by the same pathogen at a later stage of lettuce growth. Strains SHb30 (Sphingobium xenophagum), G2 (Aspergillus flavus), and Chito13 (Mycolicibacterium fortuitum) decreased seed damping-off at a better rate than a propamocarb fungicide and a Pseudomonas chlororaphis registered biocontrol agent did. In root rot bioassays, lettuce mortality was prevented by applying strains G2 and Chito13, which were at least as efficacious as the fungicide or biopesticide controls. Lettuce disease symptoms and mortality were eradicated by strain SHb30 in the first bioassay, but not in the second one. These results show that aquaponic systems are promising sources of original biocontrol agents, and that HTS-guided strategies could represent interesting approaches to identify new biocontrol agents.

[Strabismus and reading: Effect of strabismus on reading tests in children from 8 to 11 years]. / Strabisme et lecture : incidence du strabisme sur des tests de lecture chez des enfants de 8 à 11 ans.

PURPOSE: We try to show a relationship between strabismus and changes in reading skills. MATERIAL AND METHODS: We have carried out a prospective study including 135 children from 8 to 11 years (French level CE2 to CM2). They were given an ophthalmologic and orthoptic examination and then divided into 4 groups: strabismus with vertical deviation without binocular vision, accommodative strabismus with binocular vision, accommodative strabismus without binocular vision and control group (children without strabismus). Each child took 4 validated reading tests: reading fluency, uncommon words reading, comparison of letters sequences without signification, searching "verbal index". RESULTS: Results are significantly lower in children with accommodative strabismus without binocular vision for two tests (reading fluency and uncommon words reading). In contrast, results for the two other tests do not differ significantly between the 4 groups. CONCLUSION: Our study demonstrated lowered reading skills in tests of reading fluency in children with accommodative strabismus without binocular vision.

Chemotherapy-driven dysbiosis in the intestinal microbiome.

BACKGROUND: Chemotherapy is commonly used as myeloablative conditioning treatment to prepare patients for haematopoietic stem cell transplantation (HSCT). Chemotherapy leads to several side effects, with gastrointestinal (GI) mucositis being one of the most frequent. Current models of GI mucositis pathophysiology are generally silent on the role of the intestinal microbiome. AIM: To identify functional mechanisms by which the intestinal microbiome may play a key role in the pathophysiology of GI mucositis, we applied high-throughput DNA-sequencing analysis to identify microbes and microbial functions that are modulated following chemotherapy. METHODS: We amplified and sequenced 16S rRNA genes from faecal samples before and after chemotherapy in 28 patients with non-Hodgkin's lymphoma who received the same myeloablative conditioning regimen and no other concomitant therapy such as antibiotics. RESULTS: We found that faecal samples collected after chemotherapy exhibited significant decreases in abundances of Firmicutes (P = 0.0002) and Actinobacteria (P = 0.002) and significant increases in abundances of Proteobacteria (P = 0.0002) compared to samples collected before chemotherapy. Following chemotherapy, patients had reduced capacity for nucleotide metabolism (P = 0.0001), energy metabolism (P = 0.001), metabolism of cofactors and vitamins (P = 0.006), and increased capacity for glycan metabolism (P = 0.0002), signal transduction (P = 0.0002) and xenobiotics biodegradation (P = 0.002). CONCLUSIONS: Our study identifies a severe compositional and functional imbalance in the gut microbial community associated with chemotherapy-induced GI mucositis. The functional pathways implicated in our analysis suggest potential directions for the development of intestinal microbiome-targeted interventions in cancer patients.

Combination of genomic and proteomic approaches to characterize the symbiotic population of the banana aphid (Hemiptera: Aphididae).

Aphids are known to live in symbiosis with specific bacteria called endosymbionts that have positive or negative impacts on their hosts. In this study, six banana aphid (Pentalonia nigronervosa Coquerel) strains from various geographical origins (Gabon, Madagascar, and Burundi) were screened to determine their symbiotic content, using complementary genomic (16S rDNA sequencing and specific polymerase chain reaction) and proteomic (two-dimensional difference gel electrophoresis coupled with protein identification by matrix-assisted laser desorption-ionization time-of-flight mass spectrometry) approaches. Despite the geographical heterogeneity, the combined methods allowed us to identify the same two symbionts in the six aphids strains tested: Buchnera aphidicola and Wolbachia. Although B. aphidicola is found in almost all aphid species, the systematic presence of Wolbachia in banana aphids is particularly interesting, as this bacterium usually has a low prevalence in aphid species. Phylogenetic analyses showed that the Wolbachia sp. strain found in P. nigronervosa was very similar to the strain present in aphids of the genus Cinara, known to have developed a strong and long-term symbiotic association with Wolbachia. The high level of asexual reproduction in P. nigronervosa could be linked to the presence of Wolbachia, but its prevalence also suggests that this symbiotic bacterium could play a more essential role in its aphid host.

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.

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.

Comparison of different techniques for inoculation of "Candidatus Phytoplasma mali" on apple and periwinkle in biological indexing procedure.

As phytoplasmas are non cultivable micro-organisms, the research on phytoplasmal diseases can only be achieved with infected hosts. Biological indexing (by grafting) is the simplest detection method for phytoplasmal diseases. We tested four different grafting techniques for inoculation of apple trees or periwinkles in greenhouse, including whip graft, bark graft, budding and chip-budding. All techniques were tested on apple trees (six trees per phytoplasma isolates) in insect-proof greenhouse. The whip and bark grafting were not feasible for periwinkle plants, because of fineness and fragility of their tissues: only the chip-budding was performed (four plants per isolate). In apple trees, the best and soonest positive results were obtained by chip and bark grafting. Except for seven transplants not-grown after grafting, 100% efficiency of inoculation was obtained by both methods. Nevertheless, the transmission of phytoplasma from transplant not-grown to rootstock was sometimes recorded (28.6%). The earliest phytoplasma symptoms after whip or bark grafting appeared after 3 months. Symptoms were obtained much later with budding and chip-budding. In case of periwinkles, infected apple and periwinkle materials were used as inoculum sources. Transmission of phytoplasma from periwinkle to periwinkle was successfully carried out by chip-budding grafting. The symptoms were observed during the second month after inoculation. The transmission of phytoplasma from infected apple material to periwinkle (by chip-budding) was achieved for 60 % of the tested samples. Moreover, the latency period before symptom observation was longer. Finally, we perceived the apple trees are more convenient and rapid than periwinkle plants for biological indexing of apple materials.

Molecular features of new Peach Latent Mosaic Viroid variants suggest that recombination may have contributed to the evolution of this infectious RNA.

Nucleotide sequences of a broad range of Peach Latent Mosaic Viroid (PLMVd) variants were determined. The variants were isolated from peach, pear, and almond tree samples collected in Tunisia. Sequence analysis confirmed the high variability of PLMVd, as no less than 119 new variants were identified. Variations included new polymorphic positions, insertions of 11 to 14 nucleotides, and new mutations within the hammerhead self-cleavage motifs. We provide the first covariation-based evidence for certain stems within the proposed secondary structure. Our covariation analysis also strengthens the view that a pseudoknot closes the replication domain. On the basis of phylogenetic tree studies and informative positions, PLMVd variants are proposed to cluster into groups and subgroups likely to have resulted from recombination events. PLMVd thus emerges as a suitable viroid for retracing the evolution of an RNA genome.

Establishment of a new method for rapid and precise estimation of apple proliferation phytoplasma concentration in periwinkle.

Quantification of a plant pathogen is essential to study its population dynamic in various conditions and to relate symptom expression with pathogen concentration. Up to now, very few methods have been published to quantify phytoplasmas. So, the objective of this work was to establish a method able to quantify the Apple Proliferation (AP) phytoplasma populations in periwinkles. The present work was based on a method previously published to detect AP phytoplasma. This method was optimized to transform it into a quantitative method. First, a new probe specific for AP detection was applied. This probe successfully detected only AP isolates (versus closely related ESFY and PD phytoplasmas). Secondly, the method was adapted to allow the quantification of phytoplasma in periwinkle leaves. For quantification, the calibration curve was built on serial dilutions of a plasmid containing the amplified fragment (phytoplasma 16Sr gene). The limit of detection of the method was one copy of cloned phytoplasma DNA in the reaction while the lower and upper limits of quantification were 102 and 108. Sample DNA extracts were diluted 100X before amplification and standards were prepared in 100x diluted DNA extract from healthy plant. Using the calibration curve, the concentrations in the tested samples were calculated at 2 x 10(5) to 10(6) individuals per mg of fresh midrib. This work is a preliminary step to study the interaction of phytoplasmas with their hosts in relation to symptoms expression.

Biocontrol of blue mold on apple fruits by Aureobasidium pullulans (strain Ach 1-1): in vitro and in situ evidence for the possible involvement of competition for nutrients.

Aureobasidium pullulans strain Ach1-1 was recently isolated for its biocontrol effectiveness against Penicillium expansum, the causal agent of blue mold on harvested apples. In the present study, strain Ach1-1 was found to be very effective in controlling P. expansum on apple wounds. For in vitro tests, strain Ach1-1 and P. expansum were cocultured in the presence of apple juice (0 - 5%) using a system preventing direct contact between both agents. The presence of the antagonist greatly reduced germination of conidia at low (0.1, 0.5 and 1%) but not at high (5%) juice concentrations. Germination of previously inhibited conidia at 0.5% apple juice was partially restored in the presence of the antagonist when fresh juice was added at a final concentration of 5%, and completely recovered at both 0.5 and 5% juice concentrations in the absence of the antagonist. These data show that P. expansum conidia are able to germinate when cocultered with strain Ach1-1 in conditions of sufficient rather than limited nutrient availability and that the antagonist does not affect the viability of these conidia, indicating that the inhibitory effect of strain Ach1-1 on conidia germination may be due to a competition for nutrients. Such observation was confirmed in situ since the application of high amounts of exogenous amino acids, vitamins or sugars on apple wounds significantly reduced the protective level of strain Ach1-1 against P. expansum, the most important effect was obtained with amino acids followed by vitamins and then by sugars. The present work provides both in vitro and in situ evidence that the biocontrol activity of strain Ach1-1 against P. expansum essentially relies on competition for apple fruit nutrients, especially amino acids.

Genomic structure of new Tunisian peach latent mosaic viroid variants.

Peach latent mosaic viroid (PLMVd) is a single-stranded circular RNA that do not code for proteins and ranges in size from 335 to 351 nucleotides. It mainly infects peach. In this study, the sequence of 20 complete cDNA clones derived from seven PLMVd isolates detected in five Tunisian peach cultivars was analysed in 3 steps: primary structure, phylogeny and secondary structure. The analysis of the primary structure revealed that all the 20 cDNA clones sequences corresponded to different variants. They ranged in size from 336 to 341 nt. Sequence alignment of our variants with reference sequences revealed 81 polymorphic positions. Among them, 15 were never described in the literature so far. The variable positions are scattered all around the RNA molecules, but the majority of them were concentrated in the region corresponding to nucleotides 1 to 70 and 170 to 346 in the alignment. Sequence homologies between variants of the same isolate or variants of different isolates ranged from 96% to 100%. This confirms that a PLMVd isolate is composed by a complex mixture of closely related molecules. Moreover, some variants isolated from different cultivars were found to be similar, indicating that a sequence is not exclusive to a cultivar. Phylogenetic analysis of our sequences allowed their clustering into two groups: group I (16 variants) and group II (4 variants) that differed by 18 polymorphic positions. Further phylogenetic analysis and sequence alignment of our sequences and the reference sequences were done. It revealed that our sequences were similar to the reference sequence Hd8 in the regions delimited by nucleotides 1 to 69 (region 1) and 268 to 343 (region 5) and to the reference sequence Hd6 in the region between nucleotides 150 and 200 (region 3). The other regions corresponding to nucleotides 70 to 149 (region 2) and 201 to 267 (region 4) were similar for all the sequences. These observations revealed that our Tunisian PLMVd variants correspond to a new population never reported in the literature. Analysis of the secondary structure confirmed that all PLMVd Tunisian variants presented a branched secondary structure and revealed a new potential pseudoknot-like interaction between two loops.

High level of genetic diversity among spelt germplasm revealed by microsatellite markers.

The genetic diversity of spelt (Triticum aestivum (L.) Thell. subsp. spelta (L.) Thell.) cultivated presently is very narrow. Although the germplasm collections of spelt are extensive, the related genetic knowledge is often lacking and makes their use for genetic improvement difficult. The genetic diversity and structure of the spelt gene pool held in gene banks was determined using 19 simple sequence repeat (SSR) markers applied to 170 spelt accessions collected from 27 countries and 4 continents. The genetic distances (1 - proportion of shared alleles) were calculated and an unweighted pair-group method with arithmetic averaging (UPGMA)-based dendrogram was generated. The genetic diversity was high: 259 alleles were found and the mean interaccession genetic distance was 0.782 +/- 0.141. The dendrogram demonstrated the much higher genetic diversity of spelt held in germplasm collections than in the currently used genotypes. Accessions with the same geographical origin often tended to cluster together. Those from the Middle East were isolated first. All but one of the Spanish accessions were found in a unique subcluster. Most accessions from eastern Europe clustered together, while those from northwestern Europe were divided into two subclusters. The accessions from Africa and North America were not separated from the European ones. This analysis demonstrates the extent of genetic diversity of spelts held in germplasm collections and should help to widen the genetic basis of cultivated spelt in future breeding programs.

In situ development and application of cDNA-AFLP to isolate genes of Candida oleophila (strain O) potentially involved in antagonistic properties against Botrytis cinerea.

The yeast Candida oleophila (strain O) presents a high level of protective activity against Botrytis cinerea (gray mold) on postharvest apples. The cDNA-AFLP technique allows the comparison of mRNA populations extracted from cells grown in different conditions. In order to isolate yeast genes potentially involved in biological control properties, that technique was applied on strain O cells growing on apple wounds. The biological control properties of 8 C. oleophila strains and strain O were assessed in order to compare the gene expression of a non antagonistic strain against gene expression of strain O. In the absence of a non-antagonistic strain, an other comparison model was designed. It was based on the growth of strain O in different in situ conditions: strain O applied on apple wounds (O), strain O applied on apple wounds in presence of B. cinerea (B) and B. cinerea alone on apple wounds (F). A recovering technique, based on the washing of cells in the wound and a RNA extraction method followed by a DNase treatment were optimised before cDNA-AFLP application. Thirteen primer pairs were used. Their application resulted in an average of 54 and 55 bands for O and B respectively whereas no bands were observed for F. Among these bands, 8 were expressed more intensely in presence of the pathogen (1.1% of the fragments).

Feed restriction in young bulls alters the onset of puberty in relationship with plasma insulin-like growth factor-I (IGF-I) and IGF-binding proteins.

The objectives of this study were to evaluate the effect of feed restriction and re-alimentation on the onset of puberty and IGF status in peripubertal male calves and to compare the radioimmunoassay (RIA) and western ligand blotting (WLB) methods for bovine IGFBP-2. Twelve prepubertal 290 d-old Belgian Blue bulls (mean weight: +/- 290 kg) were randomly assigned in three groups: a control group (NG; n = 4) receiving a classic fattening diet to induce "normal" growth (1.48 kg/d), a feed restricted group (RG; n = 4) to obtain reduced growth (0.50 kg/d) and, a severely restricted group (SG; n = 4) to nearly stop growth (0.08 kg/d). The feed restriction period was maintained over a period of 114 d. After the period of differential feeding, all animals received the control feed regime over a period of 100 d. Blood samples were collected at fortnightly intervals. Circulating IGF-I was measured by RIA whereas plasma IGFBPs was evaluated by WLB; IGFBP-2 was additionally quantified by RIA procedure. At the beginning of the trial, IGF-I levels were low (<100 ng/ml) and similar in the three groups in accordance with prepubertal status. In the NG group, a progressive rise in IGF-I was observed from Day 42 to Day 142 whereas in the RG and SG groups, IGF-I levels did not change until the experimental restriction period ended. The delay of the rise in plasma IGF-I was longer for the SG group, IGF-I remained low until 2 wk after the end of the period of restricted feeding. Surprisingly, although differences were detected for IGF-I levels between the three groups, the IGFBP-2 and -3 data, evaluated by WLB could only discriminate between NG and SG group and not between NG and RG. However, by using a RIA method, an IGFBP-2 decrease was observed in the NG group coincident with increasing IGF-I levels. For both RG and SG groups, IGFBP-2 levels remained high throughout the feed restriction period whereas plasma IGFBP-2 levels declined upon feeding in both groups. During this feed restriction period, IGFBP-2 was significantly lower in NG than in RG or SG groups. Moreover, SG group animals had higher levels in plasma IGFBP-2 than RG animals. In conclusion, puberty is characterized by developmental changes in plasma IGF-I and IGFBPs that were altered by feed restriction. Moreover, RIA evaluation of plasma IGFBP-2 is able to better reflect group differences than WLB.

Relationships of growth hormone gene and milk protein polymorphisms to milk production traits in Simmental cattle.

The importance of milk proteins and the positive effect of administration of growth hormone (GH) on milk production, and the presence in some dairy cattle lines of greater GH concentrations prompted us to examine the presence of restriction fragment length polymorphism at the GH gene using the restriction enzyme TaqI and to investigate associations between this polymorphism in Simmental cows and bulls, as well as milk protein variants in Simmental cows, and milk production traits. Blood and milk were sampled from 279 Italian Simmental cows and semen was collected from 148 bulls of the same breed. Two fragment bands, denoted A and B, of 6200 and 5200 bp respectively, were examined and three patterns, AA, AB and BB, were found in both animal samples. All variants previously reported in other studies, for kappa, beta, and alpha s1-caseins, and beta-lactoglobulin, were found in the cows' samples. For the cows' samples, a BLUP (Best Linear Unbiased Predictor) analysis of results was performed using a REML (Restricted Maximum Likelihood) program and known heritabilities, whereas for bulls we have performed a General Linear Model analysis. The effect of GH gene polymorphism, using TaqI restriction enzyme, on milk production traits was not significant, but bulls of BB pattern had a higher breeding value for milk yield than AA bulls (P < 0.05). For the kappa-casein genotypic effects, cows of AB genotype gave milk with 1.53 +/- 0.70 g/kg less fat than cows of AA genotype. In addition, breeding values for milk protein content were significantly higher in BB bulls, with 0.87 +/- 0.32 and 0.71 +/- 0.34 g/kg more milk protein than AA and AB bulls respectively. Thus, our results revealed a GH gene polymorphism and indicated significant effects of milk protein polymorphisms on milk production traits in the Italian Simmental breed.

Pit-1 gene polymorphism, milk yield, and conformation traits for Italian Holstein-Friesian bulls.

The growth hormone factor-1/pituitary-specific transcription factor Pit-1 is responsible for the expression of growth hormone in mammals. Mutations in Pit-1 have been found in growth hormone disorders of mice and humans. We studied the eventual association between Pit-1 polymorphism using the HinfI enzyme and the milk yield and conformation traits of 89 Italian Holstein-Friesian bulls. A strategy employing polymerase chain reaction was used to amplify a 451-bp fragment from semen DNA. Digestion of polymerase chain reaction products with HinfI revealed two alleles: allele A was not digested (451-bp fragment), and allele B was cut at one restriction site, generating two fragments of 244 and 207 bp. Three patterns were observed; frequencies were 2.2, 31.5, and 66.3% for AA, AB, and BB, respectively. Fixed and mixed linear models were fitted on daughter yield deviations for milk yields and on deregressed proofs for conformation traits. Predictions were weighted using the inverse of the estimated variance of records. The models used contained mean and gene substitution effects for Pit-1 A allele as fixed effects and random sire effect for the mixed model. The A allele was found to be superior for milk and protein yields, inferior for fat percentage, and superior for body depth, angularity, and rear leg set, which is difficult to explain. A canonical transformation revealed that Pit-1 had three actions, one linked to milk yield traits and angularity, a second linked to body depth and rear leg set, and a third linked to lower fat yields and to higher angularity.

Relationships of polymorphisms for growth hormone and growth hormone receptor genes with milk production traits for Italian Holstein-Friesian bulls.

Allelic variation in the structural or regulatory sequences of growth hormone and its receptor genes might directly or indirectly affect milk traits. This possibility prompted us to investigate the eventual relationships of restriction fragment length polymorphisms at the locus of bovine growth hormone (using TagI and MspI restriction enzymes) and its receptor (using TaqI restriction enzyme) to PTA of milk production traits of bulls. Ninety-one Italian Holstein-Friesian bulls were used in this experiment, and data were analyzed with a fixed linear model. The restriction fragment length polymorphisms at the growth hormone locus did not affect the milk traits studied. Six restriction enzyme TaqI bands of 7.1, 6.2, 5.7, 5.4, 4.2, and 3.3 kb with nine patterns were observed after hybridization by a cDNA probe containing the coding sequences for the intracellular C-terminal part of the receptor. The effect of this polymorphism on PTA for milk protein percentage was highly significant and was favorable for the rare (6.6%) 5.7- and 5.4-kb pattern. Our results indicate that further study is needed to explain the DNA polymorphism and to obtain more definite conclusions about effects on milk traits.