White mold detection in common beans through leaf reflectance spectroscopy

ABSTRACT This study aimed to identify wavelengths based on leaf reflectance (400-1050 nm) to estimate white mold severity in common beans at different seasons. Two experiments were carried out, one during fall and another in winter. Partial Least Squares (PLS) regression was used to establish a set of wavelengths that better estimates the disease severity at a specific date. Therefore, observations were previously divided in two sub-groups. The first one (calibration) was used for model building and the second subgroup for model testing. Error measurements and correlation between measured and predicted values of disease severity index were employed to provide the best wavelengths in both seasons. The average indexes of each experiment were of 5.8% and 7.4%, which is considered low. Spectral bands ranged between blue and green, green and red, and red and infrared, being most sensitive for disease estimation. Beyond the transition ranges, other spectral regions also presented wavelengths with potential to determine the disease severity, such as red, green, and near infrared.

Density of primary and secondary epidermal laminae of equine hoof

Differences in the microscopic morphology of the hoof in forelimbs and hindlimbs of horses have been scarcely reported in the literature, especially concerning the distribution of primary and secondary epidermal laminae in the different regions. This study aimed to determine the density of primary and secondary epidermal laminae in the hoof of horses. For this, it was used fore and hindlimbs of 16 adult mixed breed horses. With a cross section 0.5 cm above the sole, it was quantified the primary epidermal laminae in the regions of the toe, and of lateral and medial quarters. Fragments with about 1cm ³ were taken from the proximal, middle and distal thirds of the hooves, in the different regions, subjected to conventional histological techniques and examined with an optical microscope. Data were statistically analyzed in relation to the fore and hindlimbs and between their various regions. The density of primary epidermal laminae varied around the hoof circumference, with greater values in the hoof toe, which gradually decreased towards the bulb of the hoof, without difference between thoracic and pelvic limbs. The average density of the secondary epidermal laminae per primary epidermal lamina does not change around the circumference of the hoof. Our findings indicated that the density of epidermal laminae is not different between fore and hindlimbs. The variation in the density of primary epidermal laminae around the hoof seems to be part of an adaptive response to different stresses in each region. A better understanding of the structural morphology contributes to a better understanding of the diagnosis, pathophysiology, and treatment of disorders that affect the hoof.

Morphometry of primary and secondary epidermal laminae in equine hoof

We studied the length of primary and secondary epidermal laminae of the toe and the lateral and medial quarters of horses, distributed into proximal, middle and distal thirds of the hooves. Eight limbs from adult crossbred horses, four females and four males, used to pull carts without pedal conditions. Fragments were taken from different regions of the hooves and subjected to conventional histological techniques. The samples were stained with hematoxylin-eosin and analyzed by light microscopy. The primary epidermal laminae were higher in the hooves of forelimbs compared to hindlimbs in the proximal and middle thirds and the regions of the medial quarter and toe. The secondary laminae were higher in forelimb of the middle third and medial quarter. Comparing the length of the epidermal laminae between hoof parts, it was seen that the primary laminae are lower in the proximal third and higher in the toe, while the secondary laminae are lower in the proximal third and medial quarter. The results suggested that the morphology of the laminae in the different regions of the hooves is influenced through the work performed by the animal, as well as through the different distribution of forces.

Regulation of epidermal tight junctions by calcium ATPases and p38

The epidermis is the upper layer of the skin and keratinocytes are its most abundant cells. Tight junctions are cell junctions located in the granular layer of the epidermis. They maintain the polarity of the cells and regulate the movement of water-soluble molecules. Epidermal tight junctions may lose their integrity when there are defects in intercellular calcium regulation. Hailey-Hailey and Darier´s disease are dominantly inherited, blistering skin diseases. Hailey-Hailey disease is caused by mutations in the ATP2C1 gene encoding a calcium/manganese ATPase SPCA1 of the Golgi apparatus. Darier´s disease is caused by mutations in the ATP2A2 gene encoding a calcium ATPase SERCA2 of the endoplasmic reticulum. p38 regulates the differentiation of keratinocytes. The overall regulation of epidermal tight junctions is not well understood. The present study examined the regulation of tight junctions in the human epidermis with a focus on calcium ATPases and p38. Skin from Hailey-Hailey and Darier´s disease patients was studied by using immunofluorescence labeling which targeted intercellular junction proteins. Transepidermal water loss was also measured. ATP2C1 gene expression was silenced in cultured keratinocytes, by siRNA, which modeled Hailey-Hailey disease. Expression of intercellular junction proteins was studied at the mRNA and protein levels. Squamous cell carcinoma and normal human keratinocytes were used as a model for impaired and normal keratinocyte differentiation, and the role of p38 isoforms alpha and delta in the regulation of intercellular junction proteins was studied. Both p38 isoforms were silenced by adenovirus cell transduction, chemical inhibitors or siRNA and keratinocyte differentiation was assessed. The results of this thesis revealed that: i.) intercellular junction proteins are expressed normally in acantholytic skin areas of patients with Hailey-Hailey or Darier´s disease but the localization of ZO-1 expanded to the stratum spinosum; ii.) tight junction proteins, claudin-1 and -4, are regulated by ATP2C1 in non-differentiating keratinocytes; and iii.) p38 delta regulates the expression of tight junction protein ZO-1 in proliferating keratinocytes and in squamous cell carcinoma derived cells. ZO-1 silencing, however, did not affect the expression of other tight junction proteins, suggesting that they are differently regulated. This thesis introduces new mechanisms involved in the regulation of tight junctions revealing new interactions. It provides novel evidence linking intracellular calcium regulation and tight junctions.

Leaf blade quantitative anatomy of sugarcane cultivars and clones

The objective of this study was to make a quantitative assess of the anatomic characteristics of leaf blade of the sugarcane cultivars RB855113, SP80-1842, SP80-1816, RB867515 and clone RB957689 presenting different sensitivity to the mixture of sodium trifloxysulfuron + ametryn herbicides. Compared to the other cultivars assessed, RB855113 cultivar, considered more sensitive to the herbicide mixture, presented relevant differences such as greater proportion of bulliform cells, greater tissue proportion in the transverse section of the leaf blade, greater stomata and trichome density on both surfaces, thinner epidermis on the adaxial surface and length of stomata on both surfaces. The external paraclinal wall of the bulliform cells was thinner than in the common epidermis cells in all the genotypes on the adaxial and abaxial surfaces. Multivariate analysis of the data on the variables considered most relevant to explain the herbicide penetration singled out the sensitive RB855113 from the other materials. Such characteristics can explain the greater penetration, and consequently, greater sensitivity of this cultivar to the sodium trifloxysulfuron + ametryn mixture.

Characterization of Leaf-Type Ferredoxin-NADP+ Oxidoreductase (FNR) Isoforms in Arabidopsis thaliana

Life on earth is based on sunlight, which is captured in chemical form by photosynthetic reactions. In the chloroplasts of plants, light reactions of photosynthesis take place at thylakoid membranes, whereas carbon assimilation reactions occur in the soluble stroma. The products of linear electron transfer (LET), highly-energetic ATP molecules, and reducing power in the form of NADPH molecules, are further used in the fixation of inorganic CO2 molecules into organic sugars. Ferredoxin-NADP+ oxidoreductase (FNR) catalyzes the last of the light reactions by transferring electrons from ferredoxin (FD) to NADP+. In addition to LET, FNR has been suggested to play a role in cyclic electron transfer (CET), which produces ATP without the accumulation of reducing equivalents. CET is proposed to occur via two putative routes, the PGR5- route and the NDH-route. In this thesis, the leaf-type FNR (LFNR) isoforms LFNR1 and LFNR2 of a model organism, Arabidopsis thaliana, were characterized. The physiological roles of LFNRs were investigated using single and double mutant plants. The viability of the single mutants indicates functionality of both isoforms, with neither appearing to play a specific role in CET. The more severe phenotype of low-temperature adapted fnr2 plants compared to both wild-type (WT) and fnr1 plants suggests a specific role for LFNR2 under unfavorable growth conditions. The more severe phenotype of the fnr1 x fnr2 (F1 generation) plants compared to single mutants reflects down-regulated photosynthetic capacity, whereas slightly higher excitation pressure indicates mild over-excitation of electron transfer chain (ETC). However, induction of CET and various photoprotective mechanisms enable adaptation of fnr1 x fnr2 plants to scarcity of LFNR. The fnr1 fnr2 plants (F2 generation), without detectable levels of LFNR, were viable only under heterotrophic conditions. Moreover, drought stress induced acceleration of the rate of P700 + re-reduction in darkness was accompanied by a concomitant up-regulation of the PGR5-route specific components, PGR5 and PGRL1, demonstrating the induction of CET via the PGR5-route. The up-regulation of relative transcriptional expression of the FD1 gene indicates that the FD1 isoform may have a specific function in CET, while no such role could be defined for either of the LFNR isoforms. Both the membrane-bound and soluble LFNR1 and LFNR2 each appear as two distinct spots after 2D-PAGE with different isoelectric points (pIs), indicating the existence of post-translational modifications (PTMs) which do not determine the membrane attachment of LFNR. The possibility of phosphorylation and glycosylation PTMs were excluded, but all four LFNR forms were shown to contain acetylated lysine residues as well as alternative N-termini. N-terminal acetylation was shown to shift the pI of both LFNRs to be more acidic. In addition, all four LFNR forms were demonstrated to interact both with FD1 and FD2 in vitro

The Allelopathic Effect of Eucalyptus Leaf Extract on Grass Forage Seed

This study aimed to evaluate the allelopathic effect of various concentrations of an aqueous extract of eucalyptus leaves on Urochloa decumbens and Panicum maximum seeds. The extract was prepared from Eucalyptus urograndis leaves that were milled and mixed with distilled water in a 1:9 milled leaves: water ratio to obtain an extract with a defined concentration of 100%. In addition, dilutions of 50%, 25% and 12.5% were prepared, and a 0% dilution was used as a control. The experiment followed a completely randomized design, with four replicates, each of 50 seeds of U. decumbens and 50 seeds of P. maximum, arranged on filter paper moistened with each concentration of extract in a Gerbox plastic box. The results demonstrated the allelopathic potential of E. urograndis aqueous extracts applied to the seeds of U. decumbens and P. maximum. The 50% and 100% concentrations of leaf extract most strongly inhibited the germination, vigor and seedling growth of U. decumbens and P. maximum. The germination speed index and the root length were the characteristics that were most affected by the potentially allelopathic substances contained in the eucalyptus extracts at all concentrations.

Effects of ester fractions from leaf epicuticular waxes of Bauhinia rufa (Steud.) Bong. and Stryphnodendron adstringens (Mart.) Coville from cerrado on the aphid Rhopalosiphum maidis (Fitch.)

(Effects of ester fractions from leaf epicuticular waxes of Bauhinia rufa (Steud.) Bong. and Stryphnodendron adstringens (Mart.) Coville from the cerrado on the aphid Rhopalosiphum maidis (Fitch.)). Ester fractions were isolated from the leaf epicuticular waxes of Bauhinia rufa (Steud.) Bong. and Stryphnodendron adstringens (Mart.) Coville collected in the Cerrado de Emas (Pirassununga, SP, Brazil). The bioassays of toxicity and reproductive index were carried out with esters from these species and the aphid Rhopalosiphum maidis (Fitch.). It was observed that the ester fractions from Bauhinia rufa and Stryphnodendron adstringens had a negative effect on the survival of Rhopalosiphum maidis. The ester fraction from Stryphnodendron adstringens also had a negative effect on the reproductive index of the aphid. These parameters decreased with the increase of ester concentration of artificial diets.

Leaf contents of nitrogen and phenolic compounds and their bearing with the herbivore damage to Tibouchina pulchra Cogn. (Melastomataceae), under the influence of air pollutants from industries of Cubatão, São Paulo

The Atlantic Forest on the slopes of Serra do Mar around Cubatão (São Paulo, Brazil) has been affected by massive emissions of pollutants from the local growing industrial complex. The effects of air pollution on the amounts of leaf nitrogen, total soluble phenols and total tannins of Tibouchina pulchra Cogn., a common species in the area of Cubatão, were investigated, as well as the possible influence of the altered parameters on the leaf area damaged by herbivores. Fully expanded leaves were collected at two sites: the valley of Pilões river (VP), characterized by a vegetation virtually not affected by air pollution and taken as a reference; and valley of Mogi river (VM), close to the core region of the industrial complex, and severely affected by air pollution. No differences were observed for any parameters between samples collected in the summer and winter in both sites. On the other hand, compared to VP, individuals growing in VM presented higher amounts of nitrogen and lower amounts of total soluble phenols and total tannins, as well as higher percentages of galls per leaf and higher leaf area lost to herbivores. Regression analysis revealed that the increase in leaf area lost to herbivores can be explained by the increase of the content of nitrogen and decrease in the contents of total soluble phenols and total tannins. Although significant, the coefficients of explanation found were low for all analyses, suggesting that other biotic or abiotic factors are likely to influence leaf attack by herbivores.

Growth and leaf demography of two Cecropia species

Allometry, growth and leaf demography of two Cecropia species, one with ant mutualist (C. glazioui) and another without it (C. hololeuca), were studied in an Atlantic Rain Forest area in the State of Rio de Janeiro, SE Brazil. Stem diameter was allometrically related to height in both species. Cecropia glazioui showed higher annual growth rates and longer internodes than C. hololeuca. Leaf phenology showed a seasonal pattern in both species, but the number of leaves on each plant was more variable along the year in C. hololeuca than in C. glazioui. Survivorship curves for leaves were intermediate between Deevey's Type I and Type II curves, with young leaves of C. glazioui showing a greater survival rate and life expectancy than those of C. hololeuca. Low variability in leaf production throughout the year and high survival rate for young leaves of C. glazioui may be characteristics related to its association with ants.

Ultrastructure of the intercellular protuberances in leaves of Paepalanthus superbus Ruhl. (Eriocaulaceae)

Mature leaves of Paepalanthus superbus exhibit intercellular protuberances between the inner periclinal walls of the epidermal and the parenchyma cells surface, as well as on the surface of the parenchyma mesophyll cells. These structures are mostly prominent around the parenchyma cells, forming a gel capsule-like structure. Histochemical tests with ruthenium red indicate the pectic nature of the intercellular deposits, with scattered lipidic inclusions as revealed by sudan IV and sudan black B. Ultrastructural analyses show a fibrillar matrix with scattered fimbriate and tubular structures, and a distinct margin delimited by a dense membrane-like structure. Our results suggest that the protuberances are derived from secretory activity, and are formed after the development of the intercellular spaces. For P. superbus this structure may represent an important cell wall specialisation, related with the adhesion and transport mechanisms between cells.

Comparative analysis of leaf cell-wall polysaccharides of Dialypetalanthus fuscescens and Bathysa meridionalis: evidence of biochemical similarities between Dialypetalanthaceae and Rubiaceae-Cinchonoideae

Dialypetalanthus fuscescens is an Amazonian endemic species with problematic taxonomic position. This neotropical rainforest tree belongs to the monospecific Dialypetalanthaceae. In the present work, we analysed the leaf cell-wall polysaccharide composition of Dialypetalanthus fuscescens and compared it to that of Bathysa meridionalis (Rubiaceae-Cinchonoideae). Glycosyl composition and glycosyl-linkage analysis indicated that both species have similar cell wall composition. Arabinogalactans were the major component of the pectic polysaccharides and xylans, although being reported in minor amounts in dicots, were found to be the predominant hemicellulosic polysaccharide in cell walls of both species. These findings are in agreement with previous data on cell wall composition reported for Rubiaceae and corroborate the current suggestion of the possible link between this family and Dialypetalanthaceae.

Anatomy and ontogenesis of hymenopteran leaf galls of Struthanthus vulgaris Mart. (Loranthaceae)

Leaves of Struthanthus vulgaris Mart. (Loranthaceae) exhibit galls induced by a Hymenoptera. These galls pass through five developmental stages. In the first stage, a small brown swelling is observed on the surface of the leaf. Internally, the chlorenchyma cells around the eggs of the gall-makers are divided. In the second stage, the gall enlarges and its surface assumes a wavy appearance with a depressed region in its center. Within this depression, an incompletely divided gall chamber with embryos is observed. Neoformed parenchyma is present around the chamber and the secondary walls of fibers and sclereids are no longer observed. The vascular parenchyma shows hyperplasia. In the third stage, the gall grows larger and adopts an ellipsoidal shape. Fissures appear on the gall epidermis and the neoformed parenchyma is conspicuous, with a cortical and a medullar region. In the medullar region, each gall chamber, with one inducer in larval phase, is lined with 1-2 layers of nutritive tissue. The gall is larger still at the fourth stage of development and a periderm coats most of the gall. New vascular bundles, sclereids, and fibers are formed. The gall-makers are in advanced larval phase and no nutritive tissue cells are observed. In the fifth stage, the gall reaches its definitive size and the inducers are in the pupa phase. At this stage, the cortical region undergoes slight hypertrophy. The senescent gall shows the orifices of the exit channel made by the adult gallmakers. The anatomical studies of the hymenopteran gall enabled to compare this gall with a dipteran one, previously discribed in the same plant host. It is suggested that during the maturation of the gall, specific key processes are triggered, which bring about a specific cecidogenesis.

Leaf anatomy variation within and between three "restinga" populations of Erythroxylum ovalifolium Peyr: (Erythroxylaceae) in Southeast Brazil

Erythroxylum ovalifolium is a woody shrub widespread in the "restinga", i.e. the open scrub vegetation of the Brazilian coastal sandy plains. We examined leaf anatomy variation of this species both within populations and between populations of three "restingas" in the state of Rio de Janeiro. Sites were ca.100 km far from each other and differed in regard to rainfall and vegetation structure: a dry, open site; a wet, dense site and an intermediate one. Microhabitats within sites were: (i) exposed to full irradiance, outside vegetation islands; (ii) partially exposed to full irradiance, at the border of vegetation islands; (iii) shaded, inside vegetation islands. Leaf anatomy parameters were measured for five leaves collected in each of five plants per microhabitat, in each population; they were thickness of the leaf blade, of the palisade and spongy parenchyma, and of the adaxial and abaxial epidermis. Leaves from the dry, open site had narrower abaxial epidermis and a smaller contribution of spongy parenchyma to total leaf blade thickeness than the other two sites, which we attributed to water stress. Adaxial epidermis and leaf are thicker in more exposed microhabitats (i and ii, above), irrespective of site. We proposed that between-site anatomical variation in traits related to water stress, and within-site anatomical variation in traits related to light-use are indicative of ecological plasticity and might help explain the high abundance of E. ovalifolium in the studied populations and along the State of Rio de Janeiro coast.

Leaf optical properties of two liana species Canavalia parviflora Benth. and Gouania virgata Reissk in different light conditions

Lianas are plants that depend on support to reach some appreciable height, and they represent an important structural component of tropical forests. Although they predominate in clearings and gaps, some species survive in the understory. Changes in irradiance between these environments can affect leaf morphology and absorption of photosynthetic active radiation (PAR). We had examined the effects of different light regimes on leaf optical properties, chlorophyll content, specific leaf area, and leaf surface morphology in young seedlings of Canavalia parviflora Benth. (Fabaceae) and Gouania virgata Reissk (Rhamnaceae). The seedlings were distributed on workbenches covered by different layers of neutral shade netting, thus creating three levels of light intensity corresponding to about 40%, 10% and 1.5% of solar irradiance. Plants growing in full sun were used as a control. Both species exhibited an increase in reflectance in full sun and alterations in leaf morphology. Reduction in irradiance induced an increase in absorptance (decrease in reflectance and transmittance) in C. parviflora leaves in the green due to higher chlorophyll content. In G. virgata the spectral leaf changes were less observable. However, the efficiency of absorption was more pronounced in G. virgata than in C. parviflora leaves under 40%, 10% and 1.5% photon flux density (PFD). The greater efficiency of absorption in G. virgata was due to a larger specific leaf area (SLA) under these conditions. The adjustments in leaf optical properties can aid these species in overall carbon gain under limited light conditions.