L'efecte del pH i del sistema de cultiu en el creixement de plantes de raves (Raphanus sativus L. )

Treball de recerca realitzat per un alumne d’ensenyament secundari i guardonat amb un Premi CIRIT per fomentar l'esperit científic del Jovent l’any 2008. Es pretén determinar quina influència exerceix el pH i el mètode de cultiu en el creixement i desenvolupament de les plantes de rave. S’han estudiat sis objectius concrets. Primerament, la influència del pH en el creixement de plantes de rave. Segonament, comparar dos sistemes de cultiu hidropònics: amb aigua i amb perlita. Tercerament, determinar la rendibilitat econòmica de cada sistema segons els costos i els resultats obtinguts. En quart lloc, avaluar la possibilitat d’emprar aquests mètodes a major escala i com alternativa al cultiu tradicional al sòl. En cinquè lloc, estudiar per microscòpia electrònica de rastreig els diferents epitelis de la planta. I, finalment, estudiar com afectaria el fenomen de la pluja àcida, mitjançant l’observació de l’efecte de pH àcids en el creixement de la planta.

Descrição da fêmea, genitália do macho e larva de Ocyptamos sativus (Curran, 1941) (Diptera: Syrphidae)

The female, male genital apparatus and larva of Ocyptamus sativus are described. A short report of the predatory habits of this species is made.

Amplification of the housekeeping sigma factor in Pseudomonas fluorescens CHA0 enhances antibiotic production and improves biocontrol abilities.

Pseudomonas fluorescens CHA0 produces a variety of secondary metabolites, in particular the antibiotics pyoluteorin and 2,4-diacetylphloroglucinol, and protects various plants from diseases caused by soilborne pathogenic fungi. The rpoD gene encoding the housekeeping sigma factor sigma 70 of P. fluorescens was sequenced. The deduced RpoD protein showed 83% identity with RpoD of Pseudomonas aeruginosa and 67% identity with RpoD of Escherichia coli. Attempts to inactivate the single chromosomal rpoD gene of strain CHA0 were unsuccessful, indicating an essential role of this gene. When rpoD was carried by an IncP vector in strain CHA0, the production of both antibiotics was increased severalfold and, in parallel, protection of cucumber against disease caused by Pythium ultimum was improved, in comparison with strain CHA0.

Pyoluteorin production by Pseudomonas fluorescens strain CHA0 is involved in the suppression of Pythium damping-off of cress but not of cucumber

Pseudomonas fluorescens strain CHA0 is an effective biocontrol agent of various soilborne pathogens. It controls damping-off or root rot caused byPythium ultimum on cucumber, wheat and cress. Strain CHA0 synthesizes several antibiotic metabolites such as hydrogen cyanide, 2,4-diacetylphloroglucinol, and pyoluteorin. The role of pyoluteorin in the suppression of damping-off was investigated. Two Tn5 mutants (CHA660 and CHA661) of strain CHA0 were isolated which had lost the capacity to produce pyoluteorin but still produced 2,4-diacteylphloroglucinol and HCN. These mutants still inhibitedP. ultimum on malt agar (which favours the production of 2,4-diacetylphloroglucinol) but had partially lost the ability to inhibit this pathogen on King's B agar (which favours the production of pyoluteorin). The two pyoluteorin-negative mutants showed a reduced capacity to suppress damping-off of cress caused byP. ultimum but were as effective in the protection of cucumber against this pathogen as the wild-type strain. These results indicate that, depending on the plant, pyoluteorin production plays a role in the suppression of damping-off by strain CHA0 without being a major mechanism in disease suppression. We suggest that the contribution of pyoluteorin to the biocontrol activity of strain CHA0 is determined by the quantity of this antibiotic produced in the rhizosphere, which might depend on the root exudates of the host plant.

Is the ability of biocontrol fluorescent pseudomonads to produce the antifungal metabolite 2,4-diacetylphloroglucinol really synonymous with higher plant protection?

The antifungal compound 2,4-diacetylphloroglucinol (Phl) contributes to biocontrol in pseudomonads, but whether or not Phl(+) biocontrol pseudomonads display higher plant-protecting activity than Phl(-) biocontrol pseudomonads remains to be demonstrated. This issue was addressed by assessing 230 biocontrol fluorescent pseudomonads selected from a collection of 3132 bacterial isolates obtained from 63 soils worldwide. One-third of the biocontrol pseudomonads were Phl(+) and almost all Phl(+) isolates also produced hydrogen cyanide (HCN). The only Phl(+) HCN(-) strain did harbor hcn genes, but with the deletion of a 134 bp hcnC fragment corresponding to an ADP-binding motif. Statistical analysis of biocontrol isolate distributions indicated that Phl production ability was associated with superior disease suppression activity in the Pythium-cucumber and Fusarium-tomato pathosystems, but this was also the case with HCN production ability. However, HCN significance was not as strong, as indicated both by the comparison of Phl(-) HCN(+) and Phl(-) HCN(-) strains and by correlation analyses. This is the first population-level demonstration of the higher plant-protecting activity of Phl(+) biocontrol pseudomonads in comparison with Phl(-) biocontrol pseudomonads.

Enhanced production of indole-3-acetic acid by a genetically modified strain of Pseudomonas fluorescens CHA0 affects root growth of cucumber but does not improve protection of the plant against Pythium root rot

The biocontrol strain CHA0 of Pseudomonas fluorescens produces small amounts of indole-3-acetic acid via the tryptophan side chain oxidase and the tryptophan transaminase pathways. A recombinant plasmid (pME3468) expressing the tryptophan monooxygenase pathway was introduced into strain CHA0; this resulted in elevated synthesis of indole-3-acetic acid in vitro, especially after addition of -tryptophan. In natural soil, strain CHA0/pME3468 increased fresh root weight of cucumber by 17-36%, compared to the effect of strain CHA0; root colonization was about 106 cells per g of root. However, both strains gave similar protection of cucumber against Pythium ultimum. In autoclaved soil, at 6×107 cells per g of root, strain CHA0 stimulated growth of roots and shoots, whereas strain CHA0/pME3468 caused root stunting and strong reduction of plant weight. These results are in agreement with the known effects of exogenous indole-3-acetic acid on plant roots and suggest that in the system examined, indole-3-acetic acid does not contribute to the biocontrol properties of strain CHA0.

Biotic Factors Affecting Expression of the 2,4-Diacetylphloroglucinol Biosynthesis Gene phlA in Pseudomonas fluorescens Biocontrol Strain CHA0 in the Rhizosphere.

ABSTRACT Production of the polyketide antimicrobial metabolite 2,4-diacetyl-phloroglucinol (DAPG) is a key factor in the biocontrol activity of Pseudomonas fluorescens CHA0. Strain CHA0 carrying a translational phlA'-'lacZ fusion was used to monitor expression of the phl biosynthetic genes in vitro and in the rhizosphere. Expression of the reporter gene accurately reflected actual production of DAPG in vitro and in planta as determined by direct extraction of the antimicrobial compound. In a gnotobiotic system containing a clay and sand-based artificial soil, reporter gene expression was significantly greater in the rhizospheres of two monocots (maize and wheat) compared with gene expression in the rhizospheres of two dicots (bean and cucumber). We observed this host genotype effect on bacterial gene expression also at the level of cultivars. Significant differences were found among six additional maize cultivars tested under gnotobiotic conditions. There was no difference between transgenic maize expressing the Bacillus thuringiensis insecticidal gene cry1Ab and the near-isogenic parent line. Plant age had a significant impact on gene expression. Using maize as a model, expression of the phlA'-'lacZ reporter gene peaked at 24 h after planting of pregerminated seedlings, and dropped to a fourth of that value within 48 h, remaining at that level throughout 22 days of plant growth. Root infection by Pythium ultimum stimulated bacterial gene expression on both cucumber and maize, and this was independent of differences in rhizosphere colonization on these host plants. To our knowledge, this is the first comprehensive evaluation of how biotic factors that commonly confront bacterial inoculants in agricultural systems (host genotype, host age, and pathogen infection) modulate the expression of key biocontrol genes for disease suppression.

Saponins from the Spanish saffron Crocus sativus are efficient adjuvants for protein-based vaccines.

Protein and peptide-based vaccines provide rigorously formulated antigens. However, these purified products are only weakly immunogenic by themselves and therefore require the addition of immunostimulatory components or adjuvants in the vaccine formulation. Various compounds derived from pathogens, minerals or plants, possess pro-inflammatory properties which allow them to act as adjuvants and contribute to the induction of an effective immune response. The results presented here demonstrate the adjuvant properties of novel saponins derived from the Spanish saffron Crocus sativus. In vivo immunization studies and tumor protection experiments unambiguously establish the value of saffron saponins as candidate adjuvants. These saponins were indeed able to increase both humoral and cellular immune responses to protein-based vaccines, ultimately providing a significant degree of protection against tumor challenge when administered in combination with a tumor antigen. This preclinical study provides an in depth immunological characterization of a new saponin as a vaccine adjuvant, and encourages its further development for use in vaccine formulations.

Impact of Pseudomonas fluorescens strain CHA0 and a derivative with improved biocontrol activity on the culturable resident bacterial community on cucumber roots

Information on the effects of released wild-type or genetically engineered bacteria on resident bacterial communities is important to assess the potential risks associated with the introduction of these organisms into agroecosystems. The rifampicin-resistant biocontrol strain Pseudomonas fluorescens CHA0-Rif and its derivative CHA0-Rif/pME3424, which has improved biocontrol activity and enhanced production of the antibiotics 2,4-diacetylphloroglucinol (Phl) and pyoluteorin (Plt), were introduced into soil microcosms and the culturable bacterial community developing on cucumber roots was investigated 10 and 52 days later. The introduction of either of the two strains led to a transiently enhanced metabolic activity of the bacterial community on glucose dimers and polymers as measured with BIOLOG GN plates, but natural succession between the two sampling dates changed the metabolic activity of the bacterial community more than did the inoculants. The introduced strains did not significantly affect the abundance of dominant genotypic groups of culturable bacteria discriminated by restriction analysis of amplified 16S rDNA of 2500 individual isolates. About 30-50% of the resident bacteria were very sensitive to Phl and Plt, but neither the wild-type nor CHA0-Rif/pME3424 changed the proportion of sensitive and resistant bacteria in situ. In microcosms with a synthetic bacterial community, both biocontrol strains reduced the population of a strain of Pseudomonas but did not affect the abundance of four other bacterial strains including two highly antibiotic-sensitive isolates. We conclude that detectable perturbations in the metabolic activity of the resident bacterial community caused by the biocontrol strain CHA0-Rif are (i) transient, (ii) similar for the genetically improved derivative CHA0-Rif/pME3424 and (iii) less pronounced than changes in the community structure during plant growth.

Deleterious impact of a virulent bacteriophage on survival and biocontrol activity of Pseudomonas fluorescens strain CHAO in natural soil.

Many biotic and abiotic factors affect the persistence and activity of beneficial pseudomonads introduced into soil to suppress plant diseases. One such factor may be the presence of virulent bacteriophages that decimate the population of the introduced bacteria, thereby reducing their beneficial effect. We have isolated a lytic bacteriophage (phi)GP100) that specifically infects the biocontrol bacterium Pseudomonas fluorescens CHA0 and some closely related Pseudomonas strains. phiGP100 was found to be a double-stranded-DNA phage with an icosahedral head, a stubby tail, and a genome size of approximately 50 kb. Replication of phiGP100 was negatively affected at temperatures higher than 25 degrees C. phiGP100 had a negative impact on the population size and the biocontrol activity of P. fluorescens strain CHA0-Rif (a rifampicin-resistant variant of CHA0) in natural soil microcosms. In the presence of phiGP100, the population size of strain CHA0-Rif in soil and on cucumber roots was reduced more than 100-fold. As a consequence, the bacterium's capacity to protect cucumber against a root disease caused by the pathogenic oomycete Pythium ultimum was entirely abolished. In contrast, the phage affected neither root colonization and nor the disease suppressive effect of a phiDGP100-resistant variant of strain CHA0-Rif. To our knowledge, this study is the first to illustrate the potential of phages to impair biocontrol performance of beneficial bacteria released into the natural soil environment.

Two novel MvaT-like global regulators control exoproduct formation and biocontrol activity in root-associated Pseudomonas fluorescens CHA0.

Pseudomonas fluorescens CHA0 protects various crop plants against root diseases caused by pathogenic fungi. Among a range of exoproducts excreted by strain CHA0, the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT) are particularly relevant to the strain's biocontrol potential. Here, we report on the characterization of MvaT and MvaV as novel regulators of biocontrol activity in strain CHA0. We establish the two proteins as further members of an emerging family of MvaT-like regulators in pseudomonads that are structurally and functionally related to the DNA-binding protein H-NS. In mvaT and mvaV in frame-deletion mutants of strain CHA0, PLT production was enhanced about four- and 1.5-fold, respectively, whereas DAPG production remained at wild-type levels. Remarkably, PLT production was increased up to 20-fold in an mvaT mvaV double mutant. DAPG biosynthesis was almost completely repressed in this mutant. The effects on antibiotic production could be confirmed by following expression of gfp-based reporter fusions to the corresponding biosynthetic genes. MvaT and MvaV also influenced levels of other exoproducts, motility, and physicochemical cell-surface properties to various extents. Compared with the wild type, mvaT and mvaV mutants had an about 20% reduced capacity (in terms of plant fresh weight) to protect cucumber from a root rot caused by Pythium ultimum. Biocontrol activity was nearly completely abolished in the double mutant Our findings indicate that MvaT and MvaV act together as further global regulatory elements in the complex network controlling expression of biocontrol traits in plant-beneficial pseudomonads.

Síntese e atividade fitotóxica de 2-fenil-6,7-exo-isopropilidenodioxi-8-oxabiciclo [3.2.1]oct-2-eno

The [3+4] cycloaddition between furan and the oxyallyl cation generated from 1-bromo-1-phenylpropan-2-one (4), resulted in the formation of 2-phenyl-8-oxabicyclo[3.2.1]oct-6-en-3-one (5) in 30% yield. This compound was further converted into 2-phenyl-6,7-exo-isopropylidenedioxi-8-oxabicyclo[3.2.1]oct-2-ene (13) in 35.4% yield. The selective effect of compound (13) and its isomer 3-phenyl-6,7-exo-isopropylidenedioxi-8-oxabicyclo[3.2.1]oct -2-ene (1a) on the radicle growth of Sorghum bicolor L. (sorghum) and Cucumis sativus L. (cucumber) were evaluated. For both plants, compound 13 showed to be more potent than its isomer 1a.

Identificação sorológica de espécies de vírus que infetam cucurbitáceas em áreas produtoras do Maranhão

Os vírus representam sérios obstáculos para o sucesso da olericultura no mundo inteiro, constituindo a identificação daqueles de maior incidência numa região, papel fundamental para o estabelecimento de estratégias de controle. Visitas de campo foram realizadas a plantios de espécies de cucurbitáceas em áreas produtoras do Maranhão e amostras foliares foram coletadas de 118 plantas com sintomas ou suspeita de sintomas de vírus, sendo 46 de abóbora (Cucurbita moschata), 30 de melancia (Citrullus lanatus), 23 de maxixe (Cucumis anguria), 13 de pepino (C. sativus) e seis de melão (C. melo). Todas as amostras foram testadas contra anti-soros específicos para os principais vírus das famílias Bromoviridae, Comoviridae e Potyviridae que infetam cucurbitáceas no Nordeste, mediante "enzyme-linked immunosorbent assay" (ELISA) indireto e dupla difusão em agar. Os resultados revelaram a identificação sorológica de Papaya ringspot vírus (PRSV) em 64,4% das amostras analisadas, seguido de Watermelon mosaic virus-2 (WMV-2) em 15,2%, Cucumber mosaic virus (CMV) em 6,8%, Squash mosaic virus (SqMV) em 3,4% e Zucchini yellow mosaic virus (ZYMV) em 3,4%. Este levantamento confirma a predominância do PRSV em espécies de cucurbitáceas cultivadas no estado do Maranhão.

Amperometric biosensor for ascorbic acid

A L-ascorbic acid biosensor based on ascorbate oxidase has been developed. The enzyme was extracted from the mesocarp of cucumber (Cucumis sativus) by using 0.05 mol L-1 phosphate buffer, pH 5.8 containing 0.5 mol L-1 NaCl. After the dialysis versus phosphate buffer 0.05 mol L-1 pH 5.8, the enzyme was immobilized onto nylon net through glutaraldehyde covalent bond. The membrane was coupled to an O2 electrode and the yielding reaction monitored by oxygen depletion at -600 mV using flow injection analysis optimized to 0.1 mol L-1 phosphate buffer pH 5.8, as the carrier solution and flow-rate of 0.5 mL min-1. The ascorbic acid calibration curve was linear from 1.2x10-4 to 1.0x10-3 mol L-1. The evaluation of biosensor lifetime leads to 500 injections. Commercial pharmaceutical samples were analyzed with the proposed method and the results were compared with those obtained by high-performance liquid chromatography (HPLC).