Quantitative determination of anti-fungal and insecticide amides in adult plants, plantlets and callus from Piper tuberculatum by reverse-phase high-performance liquid chromatography

A rapid, sensitive and reliable reverse-phase HPLC method was used for the quantitative determination of the anti-fungal and insecticide amides, dihydropiplartine (1), piplartine (2), Delta(alpha,beta)-dihydropiperine (3) and pellitorine (4) in plants in natura, in plantlets in vitro and ex vitro, and in callus of Piper tuberculatum. Well-resolved peaks were obtained with good detection response and linearity in the range of 15.0-3000 mug/mL. The plants in natura contained compounds 1-4, the plantlets ex vitro and in vitro accumulated compounds 1-2 and 1-4, respectively, while only amide 4 was found in callus. Copyright (C) 2003 John Wiley Sons, Ltd.

BINDING OF PARACOCCIDIOIDES-BRASILIENSIS TO LAMININ THROUGH SURFACE GLYCOPROTEIN GP43 LEADS TO ENHANCEMENT OF FUNGAL PATHOGENESIS

Extracellular matrix protein laminin binds specifically to yeast forms of Paracoccidioides brasiliensis and enhances adhesion of the fungus to the surface of epithelial Madin-Darby canine kidney cells in vitro. Immunoblotting of fungal extracts showed that the gp43 glycoprotein is responsible for adhesion. This was confirmed by binding assays using purified gp43, with a K-d of 3.7 nM. The coating of P. brasiliensis yeast forms with laminin before injection into hamster testicles enhanced the fungus virulence, resulting in a faster and more severe granulomatous disease. These results indicate that interaction of fungi with extracellular matrix elements may constitute a basis for the evolution of fungal infection toward regional spreading and dissemination.

Circadian rhythm of anti-fungal prenylated chromene in leaves of Piper aduncum

Leaves of Piper aduncum accumulate the anti-fungal chromenes methyl 2,2-dimethyl-2H-1-chromene-6-carboxylate (1) and methyl 2,2-dimethyl-8-(3'-methyl-2'-butenyl)-2H-1-chromene-6-carboxylate (2). The enzymatic formation of 2 from dimethylallyl diphosphate and I was investigated using cell-free extracts of the title plant. An HPLC assay for the prenylation reaction was developed and the enzyme activity measured in the protein extracts. The prenyltransferase that catalyses the transfer of the dimethylallyl group to C-2' of 1 was soluble and required dimethylallyl diphosphate as the prenyl donor. In the leaves, the biosynthesis of the prenylated chromene 2 was time-regulated and prenyltransferase activity depended upon circadian variation. Preliminary characterisation and purification experiments on the prenyltransferase from P. aduncum have been performed. Copyright (C) 2005 John Wiley & Sons, Ltd.

Study of seed-germination and etiolation of seedlings in Emilia-sonchifolia (l) dc and Emilia-fosgerbii nicolson (Asteraceae)

The germination of seeds of Emilia sonchifolia (2n) and E. fosgerbii (4n) was analysed and our results indicate that the former present light insensitive seeds and the latter germinated only under continuous white light. The germination of seeds under shade light presented no differences between both species, However under continuous white light, which maintain 70% of Pfr, the velocity of germination was greater in Emilia sonchifolia when compared to Emilia fosbergii, indicating that the threshold of 4n seeds were greater than 2n seeds. The analysis of the fluence response curves for etiolating process indicated that the process presented no differences between both species. The same was observed for the effect of the shade light in the etiolating process.

Seed dispersal and germination of Cryptocarya moschata Nees & Martius Ex Nees, Ocotea catharinensis Mez and Endlicheria paniculata (Sprengel) Macbride (Lauraceae)

Data of seed dispersal and germination of the studied species are presented, comparing both observations from the wild and nursery. Fruits were collected from Carlos Botelho State Park (24 degrees 44' to 24 degrees 03'S, 47 degrees 46' to 48 degrees 10'W), south of São Paulo State, Brazil. O. catharinensis had low germination percentage, both in the wild and in nursery, and did not tolerate seed storage at low temperature. In nursery, diaspores with removed mesocarp of E. paniculata presented greater germination than those in entire fruits. The high levels of seedling mortality beneath mother-trees of C. moschata, when in comparison to those observed to established seedlings from diaspores dispersed by muriquis (Brachyteles arachnoides E. Geoffroy 1806, Cebidae, Primates), allied to the absence of juveniles in the understory, are in accord with the escape model of Janzen-Connel. In nursery, diaspores of C. moschata dispersed by the primates had greater germination, in smaller time, than those collected from mother-trees.

Glyceraldehyde-3-phosphate dehydrogenase of Paracoccidioides brasiliensis is a cell surface protein involved in fungal adhesion to extracellular matrix proteins and interaction with cells

The pathogenic fungus Paracoccidioides brasiliensis causes paracoccidioidomycosis, a pulmonary mycosis acquired by inhalation of fungal airborne propagules, which may disseminate to several organs and tissues, leading to a severe form of the disease. Adhesion to and invasion of host cells are essential steps involved in the infection and dissemination of pathogens. Furthermore, pathogens use their surface molecules to bind to host extracellular matrix components to establish infection. Here, we report the characterization of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) of P. brasiliensis as an adhesin, which can be related to fungus adhesion and invasion. The P. brasiliensis GAPDH was overexpressed in Escherichia coli, and polyclonal antibody against this protein was obtained. By immunoelectron microscopy and Western blot analysis, GAPDH was detected in the cytoplasm and the cell wall of the yeast phase of P. brasiliensis. The recombinant GAPDH was found to bind to fibronectin, laminin, and type I collagen in ligand far-Western blot assays. of special note, the treatment of P. brasiliensis yeast cells with anti-GAPDH polyclonal antibody and the incubation of pneumocytes with the recombinant protein promoted inhibition of adherence and internalization of P. brasiliensis to those in vitro-cultured cells. These observations indicate that the cell wall-associated form of the GAPDH in P. brasiliensis could be involved in mediating binding of fungal cells to fibronectin, type I collagen, and laminin, thus contributing to the adhesion of the microorganism to host tissues and to the dissemination of infection.

Effect of fruit maturation stage and light on seed germination of Aiphanes aculeata

Despite their spiny nature, Aiphanes palms are popular as ornamentals. They can be grown in various soil conditions, but achieve fast, luxuriant growth in sheltered positions with plenty of water. Shaded conditions are the best when plants are young. The objective of this study was to evaluate the effect of fruit maturation stage and light on the germination of seeds of A. aculeata. Fruits were collected at three different maturation stages: (1) yellow, (2) red, (3) collected on the ground without the fleshy mesocarp (removed by birds and insects). The fleshy mesocarp was also removed in treatments (1) and (2). All fruits were then treated with thiram and 25 seeds per plot were seeded in trays full of sand. The treatments (2) and (3) were subdivided in two other treatments: (a) 12 h photoperiod, (b) continuous darkness. The temperature was maintained at 30 degrees C. Seed germination began 34 days after sowing. After 160 days, the final germination percentage was (1) 54,67%, (2a) 61,33%, (2b) 65,33%, (3a) 38,00% and (3b) 45,33%. The best treatment was (2) independently of the light conditions.

Weed seed-germination .3. bidens-pilosa L

The germination of seeds of Bidens pilosa L. colected at different places was analyzed by dose-response curves for induction and reversion of germination in seeds pre-incubated at 25 degrees C or 36 degrees C during 24 hours. Our results indicate that phytochrome is responsible for the control of seed germination id Bidens pilosa and the level of pre-existing active form of phytochrome (Pfr) above the threshold switch the seed to germinate in darkness.

Analysis of seed-germination in Piptadenia-Gonoacantha (Mart) MACBR (Leguminosae Mimosoideae)

Preliminary tests indicated 20-degrees-25-degrees-C as optimum temperature range to the germination of the seeds of P. gonoacantha. Seeds germinate both under light and dark conditions, although final percentage is always higher in latest condition. The transmission spectrum of the tegument was investigated and showed an enrichment of the light in far-red region which reaches the embryo. Consequently the white light inhibits partially germination. Another factor controlling germination was the water availability. Our results indicated that the excessive water available during imbibition inhibits the germination process. According to our results we propose that this species do not have a pioneer behavior and even though P. gonoacantha had been described as a riparian vegetation species, the germination process is strongly inhibited with excessive water during imbiibition process.

Pathogenicity of isolates of Metarhizium anisopliae (Metsch.) Sorokin towards the cattle tick Boophilus microplus (Can.) (Acari : Ixodidae) under laboratory conditions

The pathogenic activity of Metarhizium anisopliae on Boophilus microplus females was assessed using five fungal isolates. Groups of 8 engorged ticks collected from cattle on free pasture were inoculated by immersion for 5 seconds in a spore suspension containing 2.5 x 10(8) conidia/ml. The groups were transferred to moist chambers in which they were maintained at 27 +/- 1 degreesC for about 15 days, their mortality being determined every 2, or occasionally 3, days. The isolate that produced the best results in this assay was used to inoculate female ticks with spore suspensions containing 0, 7.5 x 105, 7.5 x 10(6), 7.5 x 10(7) and 7.5 x 10(8) conidia/ml, under the same conditions as described above. The pathogenic action of M. anisopliae on engorged females was clearly demonstrated. Isolates E9 and AM were more effective, causing high tick mortality as well as reduced oviposition. The concentration of 7.5 x 10(8) conidia/ml was the most effective, with the fungus sporulating on 91.1% of the ticks. Mean percent oviposition was highest in the control treatment and lowest in the treatment with 7.5 x 10(8) conidia/ml.

Effect of pre-imbibition and osmoconditioning on seed-germination in Phaseolus-vulgaris L. under water-stress

The effect of osmoconditioning and controlled water sowing was analysed in seeds of Phaseolus vulgaris L. cv. carioca on germination under water stress conditions. The pre-sowing in water at low temperature to limit germination resulted in the increase in the tolerance to water stress. The osmoconditioning with PEG for 48 hours increased the tolerance of bean seeds to water stress of -0.51MPa. The osmoconditioning or controlled water sowing can be used as a practice to increase the tolerance of bean seeds to water stress before planting in the field.

Effects of plant regulators and potassium nitrate, on Rangpur lime seeds germination

The effects of plant regulators and potassium nitrate were studied on 'Rangpur' lime (Citrus limonia Osbeck) seeds germination. The seeds were removed from ripe fruits, washed, dried and stored at 4 - 5 degrees C, and treated for 24 hours before sowing and the treatments were: KNO3 0,1% and 0,2%; GA(3) 50 ppm, 100 ppm and 250 ppm; GA(4+7)+phenylmethylaminepurine 100 ppm; phenylmethylaminepurine 20 ppm and control. The germination was evaluated begining 13 days after sowing. Best results were obtained with KNO3 0,2%, which proved most beneficial in enhancing percentage and germination rate.

Seed maturation and effect of temperature regime on Trema micrantha (L.) Blume seed germination

T. micrantha (L.) Blume (Ulmaceae), a common pioneer tree species in Brazil, is used in the restoration of degraded areas. The fruits are fleshy and indehiscent, with only one water impermeable seed. During the fruiting period, fruits of different colours are found at the same time on the same branch. This research aimed to correlate fruit colour with other physical indicators of seed maturity and to Verify the effect of temperature regime on seed germination. Collected fruits were separated in to green, green-red and red colour and for each of these maturation stages, size, moisture content and dry matter of both fruits and seeds were determined. Seeds were scarified with sulphuric acid and submitted to a germination test conducted at constant (20 degreesC, 30 degreesC and 40 degreesC) and alternating (20-30 degreesC, 30-40 degreesC and 20-40 degreesC) temperatures for 15 weeks. Seed germination percentage and speed were analysed after five weeks and the fmal percentage of germinated and Viable seeds after 15 weeks. Seed maturity is attained when the fruits are green-red. At this stage, moisture content was about 64% for fruits and 10% for seeds. Alternating temperature was required for seed germination and 20-30 degreesC was the best option. Most seeds had germinated after five weeks, providing mature seeds, acid scarification and alternating temperature were used.