Quantification of Cyclobutane Pyrimidine Dimers Induced by UVB Radiation in Conidia of the Fungi Aspergillus fumigatus, Aspergillus nidulans, Metarhizium acridum and Metarhizium robertsii

Conidia are responsible for reproduction, dispersal, environmental persistence and host infection of many fungal species. One of the main environmental factors that can kill and/or damage conidia is solar UV radiation. Cyclobutane pyrimidine dimers (CPD) are the major DNA photoproducts induced by UVB. We examined the conidial germination kinetics and the occurrence of CPD in DNA of conidia exposed to different doses of UVB radiation. Conidia of Aspergillus fumigatus, Aspergillus nidulans and Metarhizium acridum were exposed to UVB doses of 0.9, 1.8, 3.6 and 5.4 kJ m-2. CPD were quantified using T4 endonuclease V and alkaline agarose gel electrophoresis. Most of the doses were sublethal for all three species. Exposures to UVB delayed conidial germination and the delays were directly related both to UVB doses and CPD frequencies. The frequencies of dimers also were linear and directly proportional to the UVB doses, but the CPD yields differed among species. We also evaluated the impact of conidial pigmentation on germination and CPD induction on Metarhizium robertsii. The frequency of dimers in an albino mutant was approximately 10 times higher than of its green wild-type parent strain after exposure to a sublethal dose (1.8 kJ m-2) of UVB radiation.

A proteomic approach to identifying proteins differentially expressed in conidia and mycelium of the entomopathogenic fungus Metarhizium acridum

Metarhizium spp. is an important worldwide group of entomopathogenic fungi used as an interesting alternative to chemical insecticides in programs of agricultural pest and disease vector control. Metarhizium conidia are important in fungal propagation and also are responsible for host infection. Despite their importance, several aspects of conidial biology, including their proteome, are still unknown. We have established conidial and mycelial proteome reference maps for Metarhizium acridum using two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization-time of flight-mass spectrometry (MALDI-TOF MS). In all, 1130 +/- 102 and 1200 +/- 97 protein spots were detected in ungerminated conidia and fast-growing mycelia, respectively. Comparison of the two protein-expression profiles reveled that only 35 % of the protein spots were common to both developmental stages. Out of 94 2-DE protein spots (65 from conidia, 25 from mycelia and two common to both) analyzed using mass spectrometry, seven proteins from conidia, 15 from mycelia and one common to both stages were identified. The identified protein spots exclusive to conidia contained sequences similar to known fungal stress-protector proteins (such as heat shock proteins (HSP) and 6-phosphogluconate dehydrogenase) plus the fungal allergen Alt a 7, actin and the enzyme cobalamin-independent methionine synthase. The identified protein spots exclusive to mycelia included proteins involved in several cell housekeeping biological processes. Three proteins (HSP 90, 6-phosphogluconate dehydrogenase and allergen Alt a 7) were present in spots in conidial and mycelial gels, but they differed in their locations on the two gels. (c) 2010 The British Mycological Society. Published by Elsevier Ltd. All rights reserved.

Photodynamic Inactivation of Conidia of the Fungi Metarhizium anisopliae and Aspergillus nidulans with Methylene Blue and Toluidine Blue

Antimicrobial photodynamic treatment (PDT) is a promising method that can be used to control localized mycoses or kill fungi in the environment. A major objective of the current study was to compare the conidial photosensitization of two fungal species (Metarhizium anisopliae and Aspergillus nidulans) with methylene blue (MB) and toluidine blue (TBO) under different incubation and light conditions. Parameters examined were media, photosensitizer (PS) concentration and light source. PDT with MB and TBO resulted in an incomplete inactivation of the conidia of both fungal species. Conidial inactivation reached up to 99.7%, but none of the treatments was sufficient to achieve a 100% fungicidal effect using either MB or TBO. PDT delayed the germination of the surviving conidia. Washing the conidia to remove unbound PS before light exposure drastically reduced the photosensitization of A. nidulans. The reduction was much smaller in M. anisopliae conidia, indicating that the conidia of the two species interact differently with MB and TBO. Conidia of green and yellow M. anisopliae mutants were less affected by PDT than mutants with white and violet conidia. In contrast to what occurred in PBS, photosensitization of M. anisopliae and A. nidulans conidia was not observed when PDT was performed in potato dextrose media.

Host preference of the bean weevil Zabrotes subfasciatus

It is largely known that the range of an insect diet is mostly determined by oviposition behavior, mainly in species with endophytic larvae such as Zabrotes subfasciatus. However, the proximate factors determining host choice and the subsequent steps leading to the expansion or reduction of the host number and occasional host shifts are largely unknown. We analyzed various factors determining host preference of Z. subfasciatus through the evaluation of: (i) oviposition preference of a wild population of Z subfasciatus on the usual host (bean) and unusual hosts (lentil, chickpea and soy), and the performance of the offspring; (ii) artificial selection for increasing preference for hosts initially less frequently chosen; (iii) comparison of oviposition behavior between two different populations (reared for similar to 30 generations in beans or chickpeas, respectively); (iv) oviposition timing on usual and unusual hosts; and (v) identification of preference hierarchies. We found that when using unusual hosts, there is no correlation between performance and preference and that the preference hierarchy changes only slightly when the population passes through several generations on the less frequently accepted host. We also found a positive response to artificial selection for increasing oviposition on the less preferred host; however, when the host-choice experiment involved two varieties of the usual host, the response was faster than when the choice involved usual and unusual hosts. Finally, beetles reared on an unusual host (chickpea) for 26 generations showed similar good fitness on both usual and unusual hosts, indicating that the use of a new host does not necessarily result in the loss of performance on the original host. Nevertheless, this population showed lower fitness on the usual host than that of the original population, suggesting an underlying partial trade-off phenomenon which may contribute to a broadening of diet of this insect species.

Cytotoxic and genotoxic effects of two medicinal species of Verbenaceae

Lantana camara L. and Lippia alba (Mill.) N. E. Br. are two important species of Verbenaceae family and are commonly used in folk medicine in many countries of Central and Southern America. The aim of this study was to investigate, for the first time, the cytotoxic and genotoxic effects of aqueous extracts from leaves of both species on Lactuca sativa (lettuce) root tip meristem cells using a cytogenetic approach. Seeds of lettuce were separately treated during 72h with different concentrations of L. camara and L. alba aqueous extracts (5, 10, 20 and 30g/L). The percentage of germination, root development and cellular behavior were analyzed and the results showed that the highest concentration of aqueous extracts reduced the mitotic index, the seeds germination and the root development of lettuce. The extracts also induced chromosome aberrations and cellular death in roots cells of L. sativa. The cytogenotoxicity of L. camara and L. alba extracts was comparatively described.

Endogenous abscisic acid and protein contents during seed development of Araucaria angustifolia

This paper describes a proteome analysis and changes in endogenous abscisic acid (ABA) contents during seed development of Araucaria angustifolia (Bert.) O. Ktze. Megagametophytes and embryonic axis tissues exhibited a similar ABA variation pattern during seed development, reaching maximum values at the pre-cotyledonary stage. The embryonic axis protein content increased until the cotyledonary stage with following stabilization at mature seed. The two-dimensional electrophoresis at the torpedo developmental stage showed approximately 230 polypeptides against 340 in the mature stage. Peptide mass fingerprinting analyses identified three polypeptides, corresponding to an AtSAC4, a late embryogenesis abundant (LEA) and a storage protein, respectively.

Differential proteome analysis of mature and germinated embryos of Araucaria angustifolia

Araucaria angustifolia is an endangered Brazilian native conifer tree. The aim of the present work was to identify differentially expressed proteins between mature and germinated embryos of A. angustifolia, using one and two dimensional gel electrophoresis approaches followed by protein identification by tandem mass spectrometry. The identities of 32 differentially expressed protein spots from two dimensional gel maps were successfully determined, including proteins and enzymes involved in storage mobilization such as the vicilin-like storage protein and proteases. A label free approach, based on spectral counts, resulted in detection of 10 and 14 mature and germinated enriched proteins, respectively. Identified proteins were mainly related to energetic metabolism pathways, translational processes. oxidative stress regulation and cellular signaling. The integrated use of both strategies permitted a comprehensive protein expression overview of changes in germinated embryos in relation to matures, providing insights into the this process in a recalcitrant seed species. Applications of the data generated on the monitoring and control of in vitro somatic embryos were discussed. Published by Elsevier Ltd.

Vivipary in Alternanthera littoralis var. maritima - first record for the Amaranthaceae

This first record of vivipary in the Amaranthaceae is reported for the foredune plant Alternanthera littoralis var. maritima (July 2008, northern coast of Brazil). Vivipary is the precocious and uninterrupted growth of the seedlings while still attached to the maternal body. We propose a hypothesis for the viviparous germination of this species based on field observations. Our record adds new perspectives to the reproductive biology of the Amaranthaceae.

Storage proteins and cell wall mobilisation in seeds of Sesbania virgata (Cav.) Pers. (Leguminosae)

The endosperm of seeds of Sesbania virgata (Cav.) Pers. accumulates galactomannan as a cell wall storage polysaccharide. It is hydrolysed by three enzymes, one of them being alpha-galactosidase. A great amount of protein bodies is found in the cytoplasm of endospermic cells, which are thought to play the major role as a nitrogen reserve in this seed. The present work aimed at understanding how the production of enzymes that degrade storage compounds is controlled. We performed experiments with addition of inhibitors of transcription (actinomycin-d and alpha-amanitin) and translation (cycloheximide) during and after germination. In order to follow the performance of storage mobilisation, we measured fresh mass, protein contents and alpha-galactosidase activity. All the inhibitors tested had little effect on seed germination and seedling development. Actinomycin-d and cycloheximide provoked a slight inhibition of the storage protein degradation and concomitantly lead to an elevation of the alpha-galactosidase activity. Although alpha-amanitin showed some effect on seedling development at latter stages, it presented the former effect and did not change galactomannan degradation performance. Our data suggest that some of the proteases may be synthesised de novo, whereas alpha-galactosidase seems to be present in the endosperm cells probably as an inactive polypeptide in the protein bodies, being probably activated by proteolysis when the latter organelle is disassembled. These evidences suggest the existence of a connection between storage proteins and carbohydrates mobilisation in seeds of S. virgata, which would play a role by assuring a balanced afflux of the carbon and nitrogen to the seedling development.

Pollination biology of Syngonanthus elegans (Eriocaulaceae - Poales)

Studies on the pollination biology of Eriocaulaceae are scarce although particularly interesting because of its inclusion in the Poales, a predominantly wind-pollinated order. The pollination biology of Syngonanthus elegans (Bong.) Ruhland was studied during two annual flowering periods to test the hypothesis that insect pollination was its primary pollination system. A field study was carried out, including observations of the morphology and biology of the flowers, insect visits and pollinator behaviour. We also evaluated seed set, seed germination and seedling development for different pollination modes. Although seeds were produced by self-pollination, pollination by small insects contributed most effectively to the reproductive success of S. elegans, resulting in the greatest seed set, with the highest germination percentage and optimum seedling vigour. The. oral resources used by flower visitors were pollen and nectar that was produced by staminate and pistillate flowers. Self-pollination played a minor role and its consequence was inbreeding depression.

Callus induction and micropropagation improved by colchicine and phytoregulators in Kappaphycus alvarezii (Rhodophyta, Solieriaceae)

Tissue culture techniques were applied for micropropagation of the red alga Kappaphycus alvarezii in order to select the best strain and experimental system for in vitro culture. Five strains were tested: brown (BR), green (GR) and red (RD) tetrasporophytes, brown female gametophyte (BFG), and a strain originating from tetraspore germination (""Edison de Paula"", EP). The effects of three culture media were tested on callus formation, regeneration from explants and from callus in the three tetrasporophytic and EP strains: seawater enriched with half-strength of von Stosch`s (VS 50) and Guillard & Ryther`s (F/2 50) solutions, plus synthetic ASP 12-NTA medium, with or without gelling agent. Explants of the EP strain were treated with glycerol and the phytoregulators indole-3-acetic acid (IAA); 2,4-diclorophenoxyacetic acid (2,4-D); and benzylaminopurine (BA), alone or in combination. The effects of colchicine (0.01%) during 24, 48, 72 hours and 14 days were analyzed in the BFG and EP strains. The EP strain showed the highest percentage of explants forming callus and regeneration from explants in VS 50, indicating its high potential for micropropagation in comparison to the other strains. Regeneration from callus was very rare. Treatments with glycerol and IAA:BA (5:1 mg L(-1)) stimulated the regeneration from explants. Significant differences were observed in the percentages of regeneration of EP strain explants treated with colchicine for 14 days. Our results indicate that IAA and BA stimulated the regeneration process, and that colchicine produced explants with high potential for regeneration, being useful for improving the micropropagation of K. alvarezii.

Are tortoises important seed dispersers in Amazonian forests?

According to most studies on seed dispersal in tropical forests, mammals and birds are considered the main dispersal agents and the role played by other animal groups remains poorly explored. We investigate qualitative and quantitative components of the role played by the tortoise Chelonoidis denticulata in seed dispersal in southeastern Amazon, and the influence of seasonal variation in tortoise movement patterns on resulting seed shadows. Seed shadows produced by this tortoise were estimated by combining information on seed passage times through their digestive tract, which varied from 3 to 17 days, with a robust dataset on movements obtained from 18 adult C. denticulata monitored with radio transmitters and spoon-and-line tracking devices. A total of 4,206 seeds were found in 94 collected feces, belonging to 50 seed morphotypes of, at least, 25 plant genera. Very low rates of damage to the external structure of the ingested seeds were observed. Additionally, results of germination trials suggested that passage of seeds through C. denticulata`s digestive tract does not seem to negatively affect seed germination. The estimated seed shadows are likely to contribute significantly to the dispersal of seeds away from parent plants. During the dry season seeds were dispersed, on average, 174.1 m away from the location of fruit ingestion; during the rainy season, this mean dispersal distance increased to 276.7 m. Our results suggest that C. denticulata plays an important role in seed dispersal in Amazonian forests and highlight the influence of seasonal changes in movements on the resulting seed shadows.

Free Cyclitol, Soluble Carbohydrate and Protein Contents in Vigna unguiculata and Phaseolus vulgaris Bean Sprouts

Seeds sprouts have been used as a good source of basic nutrients and nutraceutical compounds. The high nutritional value of seeds derives from the deposition of compounds during development. However some of these molecules are used in metabolic processes like germination, which leads to a considerable variation in their concentrations once these events are completed. In this work, we investigate the levels of inositols (myo-inositol, D-pinitol and ononitol), soluble carbohydrates and proteins in cotyledons of Phaseolus vulgaris and Vigna unguiculata sprouts. Sprouting increased myo-inositol and glucose content and reduction of raffinose and ononitol was observed. The protein levels increased in P. vulgaris and decreased in V. unguiculata sprouting. The level of sucrose was maintained in both sprouts. D-Pinitol was detected only in quiescent seeds. Our results suggested that bean sprout is an important source of proteins, sucrose, glucose and myo-inositol. Additionally, bean sprouts have low levels of raffinose, an antinutritional compound.

Population biology and regeneration of forbs and shrubs after fire in Brazilian Campos grasslands

Fire is an important factor in several ecosystems, affecting plant population biology. Campos grasslands are under constant influence of disturbance, mostly grazing and fire. However, few studies evaluated the effect of fire on plant population biology of grassland species. Therefore, we aim to analyze the effect of fire on the population biology of four species, from different functional groups and regeneration strategies: Chaptalia runcinata (forb, resprouter, absence of belowground organ), Vernonia flexuosa (forb, resprouter, presence of rhizophore), Eupatorium ligulaefolium (shrub, resprouter, presence of xylopodium) and Heterothalamus psiadioides (shrub, obligate seeder). Seven plots were established in different sites in southern Brazil: frequently burned (FB) and excluded from fire since 6 years (E). All plots were subjected to controlled burns during summer. Before experiments, populations were sampled. Further observations were carried out after 90 and after 360 days of fire experiments. In addition, we counted the number of seedlings and resprouters recruited after fire. Heat shock experiments were conducted with two species (H. psiadioides and V. flexuosa), as well as the study of the bud bank of the following species: E. ligulaefolium and V. flexuosa. The obligate seeder species had all individuals killed by fire and established only after 1 year. Resprouters, however, showed new stems immediately after fire. E. ligulaefolium and V. flexuosa showed only vegetative regeneration from belowground organs and more individuals in excluded sites 1 year after the fire. The bud bank of E. ligulaefolium tended to be larger in excluded sites, whilst V. flexuosa showed an opposite result. High temperatures did not enhance nor kill seeds from both studied species. Vegetative regeneration was the most important strategy for all studied species, except for H. psiadioides, the obligate seeder species. Fire thus, plays an important role on population structure and demography, being also important for plant recruitment.

FIRE INTENSITY AND SEVERITY IN BRAZILIAN CAMPOS GRASSLANDS

Brazilian Campos grasslands are rich in species and the maintenance of its diversity and physiognomy is dependent on disturbance (e.g. fire and grazing) Nevertheless, studies about fire intensity and severity are inexistent. The present paper describes fire parameters, using 14 experimental burn plots in southern Brazil (30 degrees 02` to 30 degrees 04`S, and 51 degrees 06` to 51 degrees 09`W. 311masl). Two sites under different fire histories were chosen: frequently burned and excluded since six years. Experimental burning was performed during summer (2006-2007), when most burning takes place in these grasslands. The following parameters were measured: air temperature and moisture, vegetation height, wind speed, fuel (fine, coarse), fuel moisture, fire temperatures (soil level and at 50cm), ash, residuals, flame freight, fire duration: burning efficiency and fire intensity were later calculated. Fuel load varied from 0.39 to 1.44kg.m(-2). and correlated positively with both fire temperature and fire intensity. Fire temperatures ranged 47 to 537.5 degrees C. being higher in the excluded site Fire intensity was low compared to grassland elsewhere (36 5-319.5kW.m(-1)), differing significantly between sties Fine fuel was the variable that best explained fire intensity. The results on fire intensity and severity in Campos grasslands can be considered a pilot study, since plots were very small. However the data provided can help other researchers to get permission for experimentation using larger plots The results provide support for further studies about the effects of fire on grassland vegetation and for studies involving fire models and fire risk prediction