Arbuscular mycorrhizal fungi alter the content and composition of secondary metabolites in Bituminaria bituminosa L.

Secondary metabolites may be affected by arbuscular mycorrhizal fungi (AMF), which are beneficial symbionts associated with the roots of most plant species. Bituminaria bituminosa (L.) C.H.Stirt is known as a source of several phytochemicals and therefore used in folk medicine as a vulnerary, cicatrising, disinfectant agent. Characteristic metabolites found in B. bituminosa are furanocoumarins and pterocarpans, which are used in cosmetics and as chemotherapeutic agents. Here we address the question whether AMF inoculation might affect positively the synthesis of these phytochemicals. B. bituminosa plants were inoculated with different AMF and several metabolites were assessed during full vegetative stage and flowering phase. Pigments (chlorophylls and carotenoids), polyphenols and flavonoids were spectrophotometrically determined; specific isoflavones (genistein), furanocoumarins (psoralene and angelicin), pterocarpans (bitucarpin A and erybraedin C) and plicatin B were assessed with HPLC; leaf volatile organic compounds were analysed using SPME and identified by GC-MS. During the vegetative stage, the inoculated plants had a high amount of furanocoumarins (angelicin and psoralen) and pterocarpans (erybraedin C and bitucarpin A). The analysis of volatile organic compounds of inoculated plants showed different chemical composition compared with non-mycorrhizal plants. Given the important potential role played by furanocoumarins and pterocarpans in the pharmaceutical industry, AMF inoculation of B. bituminosa plants may represent a suitable biotechnological tool to obtain higher amounts of such metabolites for pharmaceutical and medicinal purposes.

Nutraceutical value and safety of tomato fruits produced by mycorrhizal plants.

Tomato fruit has assumed the status of 'functional food' due to the association between its consumption and a reduced likelihood of certain types of cancers and CVD. The nutraceutical value of tomatoes can be affected by the cultivation conditions, e.g. the phytochemical content of the fruits may increase with the establishment of beneficial mycorrhizal symbioses in the plants. A multidisciplinary study was carried out to gain knowledge on the antioxidant, oestrogenic/anti-oestrogenic and genotoxic activity of tomato fruits produced by mycorrhizal plants. The present results showed that the symbiosis positively affected the growth and mineral nutrient content of tomato plants and enhanced the nutritional and nutraceutical value of tomato fruits through modifications of plant secondary metabolism, which led to increased levels of lycopene in fruits obtained from mycorrhizal plants, compared with controls. Moreover, such changes did not result in the production of mutagenic compounds, since tomato extracts induced no in vitro genotoxic effects. Fruit extracts, both hydrophilic and the lipophilic fractions, originating from mycorrhizal plants strongly inhibited 17-ß-oestradiol-human oestrogen receptor binding, showing significantly higher anti-oestrogenic power compared with controls. The present study shows that beneficial plant symbionts, such as mycorrhizal fungi, can lead to the production of safe and high-quality food, which is an important societal issue strongly demanded by both consumers and producers.

Impact of Bt corn on rhizospheric and soil eubacterial communities and on beneficial mycorrhizal symbiosis in experimental microcosms.

A polyphasic approach has been developed to gain knowledge of suitable key indicators for the evaluation of environmental impact of genetically modified Bt 11 and Bt 176 corn lines on soil ecosystems. We assessed the effects of Bt corn (which constitutively expresses the insecticidal toxin from Bacillus thuringiensis, encoded by the truncated Cry1Ab gene) and non-Bt corn plants and their residues on rhizospheric and bulk soil eubacterial communities by means of denaturing gradient gel electrophoresis analyses of 16S rRNA genes, on the nontarget mycorrhizal symbiont Glomus mosseae, and on soil respiration. Microcosm experiments showed differences in rhizospheric eubacterial communities associated with the three corn lines and a significantly lower level of mycorrhizal colonization in Bt 176 corn roots. In greenhouse experiments, differences between Bt and non-Bt corn plants were detected in rhizospheric eubacterial communities (both total and active), in culturable rhizospheric heterotrophic bacteria, and in mycorrhizal colonization. Plant residues of transgenic plants, plowed under at harvest and kept mixed with soil for up to 4 months, affected soil respiration, bacterial communities, and mycorrhizal establishment by indigenous endophytes. The multimodal approach utilized in our work may be applied in long-term field studies aimed at monitoring the real hazard of genetically modified crops and their residues on nontarget soil microbial communities.

Chemotropism in the arbuscular mycorrhizal fungus Glomus mosseae.

In this work, we report the occurrence of chemotropism in the arbuscular mycorrhizal (AM) fungus Glomus mosseae. Fungal hyphae were able to respond to host-derived signals by reorienting their growth towards roots and to perceive chemotropic signals at a distance of at least 910 microm from roots. In order to reach the source of chemotropic signals, hyphal tips crossed interposed membranes emerging within 1 mm from roots, eventually establishing mycorrhizal symbiosis. The specificity of chemotropic growth was evidenced by hyphal growth reorientation and membrane penetration occurring only in experimental systems set up with host plants. Since pre-symbiotic growth is a critical stage in the life cycle of obligate AM fungal symbionts, chemotropic guidance may represent an important mechanism functional to host root location, appressorium formation and symbiosis establishment.

The antifungal Dm-AMP1 protein from Dahlia merckii expressed in Solanum melongena is released in root exudates and differentially affects pathogenic fungi and mycorrhizal symbiosis.

• Transformed aubergine plants constitutively expressing the Dm-AMP1 antimicrobial defensin (from Dahlia merckii) were generated and characterized. • Transgenic plants were selected on kanamycin and screened by polymerase chain reaction analysis. The expression of Dm-AMP1 in plant tissues and its release in root exudates were detected by Western blot analyses. Dm-AMP1 localization was performed by immunohistochemical experiments. • Dm-AMP1 expression ranged from 0.2% to 0.48% of total soluble proteins in primary transformants and from 0.16% to 0.66% in F2 plants. Transformed clones showed resistance to the pathogenic fungus Botrytis cinerea, whose development on leaves was reduced by 36-100%, with respect to controls. The protein was released in root exudates of the transformed plants and was active in reducing the growth of the co-cultured pathogenic fungus Verticillium albo-atrum, whereas it did not interfere with recognition responses and symbiosis establishment by the arbuscular mycorrhizal fungus Glomus mosseae. • Dm-AMP1 transformants may represent a useful model to study the interactions between genetically modified plants and pathogenic fungi or beneficial nontarget microorganisms.

La audiometría tonal como método de despistaje auditivo en pacientes asintomáticos / Tonal audiometry as a method of auditive trial in asymptomatic patients

El objetivo primordial de la investigación fue determinar la utilidad de la Audiometría Tonal vía aérea y vía ósea y de la logoaudiometría como método efectivo de Despistaje Auditivo en pacientes no tratados previamente. Se presentan los resultados obtenidos al evaluar 3.148 casos de pacientes estudiados en el Departamento de Evaluación Médica Preventiva del Hospital de Clínicas Caracas y se analiza la presencia de Hipoacusia en el grupo. Se le realizó a cada uno de los pacientes la Audiometría Tonal vía aérea y logoaudiometría, y en los casos con alteración del audiograma se les realizó la vía ósea. Después de obtenidos los resultados, se analizaron y se demostró la utilidad de la Audiometría Tonal como método para despistar patologías auditivas en pacientes que no han sido tratados previamente. Para la recolección de los datos se tomó en cuenta el grado y tipo de hipoacusia según las escalas convencionales, hipoacusia leve, moderada, moderada severa, severa y profunda; conductiva, neurosensorial y mixta; y bilateral y unilateral. Se concluyó que: 1) La Audiometría Tonal es un método eficiente para detectar audición e hipoacusia; 2) El uso de la Audiometría Tonal permitió detectar la presencia de hipoacusia desde leve hasta profunda en personas asintomáticas; 3)Según el grado de hipoacusia la más observada fue la hipocausia leve; 4) Según el tipo de hipoacusia la más observada fue la hipoacusia neurosensorial; 5) La incidencia de hipoacusia es mayor en el sexo masculino que en el femenino; 6) El grado de hipoacusia más observado en ambos sexos fue la hipoacusia leve y el tipo más observado fue la neurosensorial; 7) El grado de hipoacusia resultó proporcional a la edad del paciente evaluado; 8)La audiometría Tonal es un instrumento importantísimo como método de despistaje auditivo en pacientes que acuden a chequeo médico tutorial; 9) El 55 por ciento de los casos presentó Hipoacusia bilateral y el 45 por ciento Hipoacusia Unilateral

Adhesion to hyphal matrix and antifungal activity of Pseudomonas strains isolated from Tuber borchii ascocarps.

Pseudomonas spp. isolates from Tuber borchii ascocarps, known to be able to produce phytoregulatory and biocontrol substances in pure culture, were used to perform studies on their possible physiological role in nature. Antimycotic activity was confirmed against fungal contaminants isolated from the ascocarps, suggesting that populations associated with Tuber borchii fruit bodies may play a role in the maintenance of ascocarp health. Fifty-five percent of strains tested were also able to release metabolites which affected T. borchii mycelial growth and morphogenesis in culture. On the contrary, growth of the arbuscular mycorrhizal fungus Glomus mosseae and the ectomycorrhizal fungus Laccaria bicolor, putative competitors of Tuber for mycorrhizal infection sites on roots, was not influenced by the presence of any bacterial strain. The possibility that these bacteria, which show antifungal activity and fungal growth modulation activities, might be incorporated in the developing ascocarp by means of their preferential adhesion to Tuber mycelium is discussed.

Anastomosis formation and nuclear and protoplasmic exchange in arbuscular mycorrhizal fungi.

We observed anastomosis between hyphae originating from the same spore and from different spores of the same isolate of the arbuscular mycorrhizal fungi Glomus mosseae, Glomus caledonium, and Glomus intraradices. The percentage of contacts leading to anastomosis ranged from 35 to 69% in hyphae from the same germling and from 34 to 90% in hyphae from different germlings. The number of anastomoses ranged from 0.6 to 1.3 per cm (length) of hyphae in mycelia originating from the same spore. No anastomoses were observed between hyphae from the same or different germlings of Gigaspora rosea and Scutellospora castanea; no interspecific or intergeneric hyphal fusions were observed. We monitored anastomosis formation with time-lapse and video-enhanced light microscopy. We observed complete fusion of hyphal walls and the migration of a mass of particles in both directions within the hyphal bridges. In hyphal bridges of G. caledonium, light-opaque particles moved at the speed of 1.8 +/- 0.06 microm/s. We observed nuclear migration between hyphae of the same germling and between hyphae belonging to different germlings of the same isolate of three Glomus species. Our work suggests that genetic exchange may occur through intermingling of nuclei during anastomosis formation and opens the way to studies of vegetative compatibility in natural populations of arbuscular mycorrhizal fungi.

Cellular events involved in survival of individual arbuscular mycorrhizal symbionts growing in the absence of the host

A survival strategy operating in the absence of the host was shown in obligately biotrophic arbuscular mycorrhizal (AM) symbionts. When no host-derived signals from the surrounding environment were perceived by germinating spores, fungal hyphae underwent a programmed growth arrest and resource reallocation, allowing long-term maintenance of viability and host infection capability. The early stages of mycelial growth of AM fungi were studied by a combination of time-lapse and video-enhanced light microscopy, image analysis, and immunodetection, with the aim of acquiring knowledge of cell events leading to the arrest of mycelial growth. The time-course of growth arrest was resolved by precisely timing the growth rate and magnitude of the mycelium originating from individual spores of Glomus caledonium. Extensive mycelial growth was observed during the first 15 days; thereafter, fungal hyphae showed retraction of protoplasm from the tips, with formation of retraction septa separating viable from empty hyphal segments. This active process involved migration of nuclei and cellular organelles and appeared to be functional in the ability of the fungus to survive in the absence of a host. Immunodetection of cytoskeletal proteins, metabolic activity, and the retention of infectivity of germinated spores confirmed the developmental data. The highest amounts of tubulins were detected when hyphal growth had ceased but when retraction of protoplasm was most active. This was consistent with the role of the cytoskeleton during protoplasm retraction. Succinate dehydrogenase activity in hyphae proximal to the mother spore was still detectable in 6-month-old mycelium, which remained viable and able to form appressoria and produce symbiotic structures.

Influence of arbuscular mycorrhizae and phosphate fertilization on shoot apical growth of micropropagated apple and plum rootstocks.

We studied the effects of phosphate fertilization and inoculation with the arbuscular mycorrhizal fungi Glomus mosseae (Nicol. and Gerd.) Gerdmann and Trappe, Glomus intraradices Schenck and Smith or Glomus viscosum Nicolson on shoot apical growth of plantlets that had been micropropagated from MM 106 apple (Malus pumila L.) and Mr.S. 2/5 plum (Prunus cerasifera Ehrh.) rootstocks. Unfertilized and non-mycorrhizal plantlets showed no apical growth during the post in vitro acclimation phase, whereas P fertilization induced early resumption of shoot apical growth. Growth enhancement and percentage of actively growing apices of mycorrhizal-inoculated plantlets were comparable to those obtained in plantlets fertilized with P. Furthermore, tissue P concentrations of mycorrhizal plantlets were similar to those of plantlets fertilized with P. We conclude that mycorrhizal inoculation can be used as a biotechnological tool to overcome blocked apical growth and to reduce chemical inputs, especially P inputs, to micropropagated fruit trees.

Arbuscular mycorrhizal induced changes to plant growth and root system morphology in Prunus cerasifera.

We compared root system morphogenesis of micropropogated transplants of Prunus cerasifera L. inoculated with either of the arbuscular mycorrhizal (AM) fungi Glomus mosseae or Glomus intraradices or with the ericoid mycorrhizal species Hymenoscyphus ericae. All plants were grown in sand culture, irrigated with a nutrient solution that included a soluble source of phosphorus, for 75 days after transplanting. Arbuscular mycorrhizal colonization increased both the survival and growth (by over 100%) of transplants compared with either uninoculated controls or transplants inoculated with H. ericae. Arbuscular mycorrhizal colonization increased root, stem and leaf weights, leaf area, root length and specific leaf area, and it decreased root length/leaf area ratio, root/shoot weight ratio and specific root length. Both uptake of phosphorus and its concentration in leaves were increased by AM infection, although the time course of the relationships between intensity of AM infection and P nutrition were complex and suggested a role for factors other than nutrition. The time course for the development of infection varied. It was most rapid with G. mosseae, but it was ultimately higher with G. intraradices. None of the treatments significantly affected the lengths of adventitious roots or the first-, second- or third-order laterals that developed from them. Arbuscular mycorrhizal colonization increased the intensity of branching in all root orders with the effect being most obvious on first-order lateral roots where the number of branches increased from under 100 to over 300 brances m(-1). As a result, although first-order laterals made up 55% of the root systems of control plants, the comparable value was 36% in AM-infected plants. In contrast, second-order laterals represented 25% of control root systems, but 50% of AM-colonized root systems. Glomus intraradices but not G. mosseae increased root diameter. Anatomical studies revealed no changes in the overall form of the root tip, although there were changes in the diameter of the root cap, cell numbers and cell size. Hymenoscyphus ericae increased the duration of the metaphase index. Both AM fungal treatments increased the concentrations of soluble proteins in root extracts and modified the protein profiles by the elimination and addition of protein bands detected by PAGE analysis. We conclude that AM fungal inoculation influenced processes in the root system at different levels, but not all effects were due to improved P nutrition or increased physiological age.

Effects of the antimycotic molecule Iturin A2, secreted by Bacillus subtilis strain M51, on arbuscular mycorrhizal fungi.

A new system, devised for the study of early stages of arbuscular mycorrhizal infection, was used to test the effect of the biological control agent Iturin A2, secreted by the strain M51 of Bacillus subtilis, on the development of arbuscular mycorrhizal fungi. The saprophytic growth of the fungus Glomus mosseae was inhibited by Iturin A2 concentrations higher than 100 micrograms/g of sand; whereas, in the presence of the tomato host plant, both, pre-infection events and intraradical growth were not negatively influenced by the antifungal compound; furthermore, the development of arbuscular mycorrhizal symbiosis was not impeded by the biocontrol agent in field conditions, while Fusarium oxysporum f. sp. lycopersici infection was hindered.

[Tissue necrosis: a side effect of sodium diclofenac: report of cases and discussion of the physiopathology]. / Necrose tecidual: efeito colateral do diclofenaco de sódio, relato de casos e discussão da fisiopatologia.

Diclofenac sodium is a non-steroidal anti-inflammatory widely used drug both in the treatment of the rheumatic diseases and as analgesic. The fact that is a potent analgesic spread its use to almost all fields in medicine, increasing the reports of adverse effects associated with it. The main complications reported are gastrointestinal, dermatological and of the central nervous system. The authors present seven cases that developed tissue necrosis as a result of the intramuscular injection of diclofenac sodium. Because of the low incidence or this adverse effect, a review is carried out and the physiopathologic mechanisms likely to be involved are discussed. Our aim is to warn against the indiscriminate intramuscular use of this drug.

Differential hyphal morphogenesis in arbuscular mycorrhizal fungi during pre-infection stages.

Roots of host plants elicit a local change in morphology in the hyphae of arbuscular mycorrhizal fungi, prior to the formation of appressoria. The elicited hyphae switch from their original branching pattern and apical dominance to differentiate in a new irregular, septate branching pattern with reduced inter-hyphal spacing. The extensive hyphal development associated with roots of host plants was shown to be due to the differential growth pattern described, and to precede the further cascade of events leading to appressorium formation and the development of a functional symbiosis.

Directional needle for antegrade guide wire placement with vertical arterial puncture.

A directional needle with a closed pencil-point tip and a distal side hole was developed to permit antegrade guide wire placement by way of a 90 degrees puncture angle. It has been used in over 25 patients without technical difficulties or complications. It has been very effective for catheterization of the superficial femoral artery for angioplasty, diagnostic studies, and chemotherapy infusion, providing easy antegrade access in patients in whom the traditional antegrade approach may be difficult.

Magnetic resonance imaging appearance of supernumerary soleus muscle.

A case of histologically proven supernumerary soleus muscle examined with MRI and CT is described. The diagnosis can be made using the tissue characterization properties of MRI, obviating biopsy.

Normal ventilatory movement of the right hemidiaphragm studied by ultrasonography and pneumotachography.

The ventilatory movement of the right dome of the diaphragm was studied over the range of the inspiratory capacity in 50 healthy adults in the supine position using simultaneous ultrasonography and pneumotachography. Regional differences were found in movement of the hemidiaphragm; there was greater movement of the middle and posterior thirds of the diaphragm than of the anterior third. A significant positive correlation was found between body weight and amount of diaphragm movement. An apparently greater degree of movement of the diaphragm was found in males than females; this may be related to weight. About 64% of diaphragm movement has occurred by the time midinspiratory capacity is reached. These data may help to define normal movement and also to differentiate abnormal from normal diaphragm movement.

Mutagenicity of pesticides evaluated by means of gene-conversion in Saccharomyces cerevisiae and in Aspergillus nidulans.

Nine pesticides, Afugan, Atrazine, Benomyl, Captan, Daconil, Melthaumittel, Plantvax, Saprol, and Wepsin were tested for the induction of mitotic gene-conversion in two different eucaryotic microorganisms, Saccharomyces cerevisae and Aspergillus nidulans. In S cerevisiae the pesticides were also tested after mouse liver microsomal activation; in A nidulans all tests were performed using both resting and germinating conidia. Among the tested pesticides, only Captan revealed a consistent genetic activity, three times greater than that observed with the standard mutagen methylmethansulphonate. This genetic activity of Captan is suppressed after mammalian metabolic conversion. The use of germinating conidia in A nidulans for scoring the induction of mitotic gene-conversion did not give any further information on the activity of the tested pesticides.