X-ray computed tomography to quantify tree rooting spatial distributions

Poor root development due to constraining soil conditions could be an important factor influencing health of urban trees. Therefore, there is a need for efficient techniques to analyze the spatial distribution of tree roots. An analytical procedure for describing tree rooting patterns from X-ray computed tomography (CT) data is described and illustrated. Large irregularly shaped specimens of undisturbed sandy soil were sampled from Various positions around the base of trees using field impregnation with epoxy resin, to stabilize the cohesionless soil. Cores approximately 200 mm in diameter by 500 mm in height were extracted from these specimens. These large core samples were scanned with a medical X-ray CT device, and contiguous images of soil slices (2 mm thick) were thus produced. X-ray CT images are regarded as regularly-spaced sections through the soil although they are not actual 2D sections but matrices of voxels similar to 0.5 mm x 0.5 mm x 2 mm. The images were used to generate the equivalent of horizontal root contact maps from which three-dimensional objects, assumed to be roots, were reconstructed. The resulting connected objects were used to derive indices of the spatial organization of roots, namely: root length distribution, root length density, root growth angle distribution, root spatial distribution, and branching intensity. The successive steps of the method, from sampling to generation of indices of tree root organization, are illustrated through a case study examining rooting patterns of valuable urban trees. (C) 1999 Elsevier Science B.V. All rights reserved.

Macropore sheath: quantification of plant root and soil macropore association

Plants require roots to supply water, nutrients and oxygen for growth. The spatial distribution of roots in relation to the macropore structure of the soil in which they are growing influences how effective they are at accessing these resources. A method for quantifying root-macropore associations from horizontal soil sections is illustrated using two black vertisols from the Darling Downs, Queensland, Australia. Two-dimensional digital images were obtained of the macropore structure and root distribution for an area 55 x 55 mm at a resolution of 64 mu m. The spatial distribution of roots was quantified over a range of distances using the K-function. In all specimens, roots were shown to be clustered at short distances (1-10 mm) becoming more random at longer distances. Root location in relation to macropores was estimated using the function describing the distance of each root to the nearest macropore. From this function, a summary variable, termed the macropore sheath, was defined. The macropore sheath is the distance from macropores within which 80% of roots are located. Measured root locations were compared to random simulations of root distribution to establish if there was a preferential association between roots and macropores. More roots were found in and around macropores than expected at random.

Analysis of long-term light-trap data for Helicoverpa spp. (Lepidoptera : Noctuidae) in Australia: the effect of climate and crop host plants

Regression analyses of a long series of light-trap catches at Narrabri, Australia, were used to describe the seasonal dynamics of Helicoverpa armigera (Hubner). The size of the second generation was significantly related to the size of the first generation, to winter rainfall, which had a positive effect, and to spring rainfall which had a negative effect. These variables accounted for up to 96% of the variation in size of the second generation from year to year. Rainfall and crop hosts were also important for the size of the third generation. The area and tonnage of many potential host crops were significantly correlated with winter rain. When winter rain was omitted from the analysis, the sizes of both the second and third generations could be expressed as a function of the size of the previous generation and of the areas planted to lucerne, sorghum and maize. Lucerne and maize always had positive coefficients and sorghum a negative one. We extended our analysis to catches of H. punctigera (Wallengren), which declines in abundance after the second generation. Winter rain had a positive effect on the sizes of the second and third generations, and rain in spring or early summer had a negative effect. Only the area grown to lucerne had a positive effect on abundance. Forecasts of pest levels from a few months to a few weeks in advance are discussed, along with the improved understanding of the seasonal dynamics of both species and the significance of crops in the management of insecticide resistance for H. armigera.

A protocol for the fluorometric quantification of mGFP5-ER and sGFP(S65T) in transgenic plants

The Green Fluorescent Protein (GFP) from Aequorea victor-in has begun to be used as a reporter protein in plants. It is particularly useful as GFP fluorescence can be detected in a non-destructive manner, whereas detection of enzyme-based reporters often requires destruction of the plant tissue. The use of GFP as a reporter enables transgenic plant tissues to be screened in vivo at any growth stage. Quantification of GFP in transgenic plant extracts will increase the utility of GFP as a reporter protein. We report herein the quantification of a mGFP5-ER Variant in tobacco leaf extracts by UV excitation and a sGFP(S65T) variant in sugarcane leaf and callus extracts by blue light excitation using the BioRad VersaFluor(TM) Fluorometer System or the Labsystems Fluoroskan Ascent FL equipped with a narrow band emission filter (510 +/- 5 nm). The GFP concentration in transgenic plant extracts was determined from a GFP-standard series prepared in untransformed plant extract with concentrations ranging from 0.1 to 4 mu g/ml of purified rGFP. Levels of sgfp(S65T) expression, driven by the maize ubiquitin promoter, in sugarcane calli and leaves ranged up to 0.525 mu g and 2.11 mu g sGFP(S65T) per mg of extractable protein respectively. In tobacco leaves the expression of mgfPS-ER, driven by the cauliflower mosaic virus (CaMV) 35S promoter, ranged up to 7.05 mu g mGFP5-ER per mg extractable protein.

X-ray absorption and phase contrast imaging to study the interplay between plant roots and soil structure

Plant performance is, at least partly, linked to the location of roots with respect to soil structure features and the micro-environment surrounding roots. Measurements of root distributions from intact samples, using optical microscopy and field tracings have been partially successful but are imprecise and labour-intensive. Theoretically, X-ray computed micro-tomography represents an ideal solution for non-invasive imaging of plant roots and soil structure. However, before it becomes fast enough and affordable or easily accessible, there is still a need for a diagnostic tool to investigate root/soil interplay. Here, a method for detection of undisturbed plant roots and their immediate physical environment is presented. X-ray absorption and phase contrast imaging are combined to produce projection images of soil sections from which root distributions and soil structure can be analyzed. The clarity of roots on the X-ray film is sufficient to allow manual tracing on an acetate sheet fixed over the film. In its current version, the method suffers limitations mainly related to (i) the degree of subjectivity associated with manual tracing and (ii) the difficulty of separating live and dead roots. The method represents a simple and relatively inexpensive way to detect and quantify roots from intact samples and has scope for further improvements. In this paper, the main steps of the method, sampling, image acquisition and image processing are documented. The potential use of the method in an agronomic perspective is illustrated using surface and sub-surface soil samples from a controlled wheat trial. Quantitative characterization of root attributes, e.g. radius, length density, branching intensity and the complex interplay between roots and soil structure, is presented and discussed.

Does maintaining green leaf area in sorghum improve yield under drought? I. Leaf growth and senescence

Production of sorghum [Sorghum bicolor (L.) Moench], an important cereal crop in semiarid regions of the world, is often limited by drought. When water is limiting during the grain-filling period, hybrids possessing the stay-green trait maintain more photosynthetically active leaves than hybrids not possessing this trait. To improve yield under drought, knowledge of the extent of genetic variation in green leaf area retention is required. Field studies were undertaken in north-eastern Australia on a cracking and self-mulching gray clay to determine the effects of water regime and hybrid on the components of green leaf area at maturity (GLAM). Nine hybrids varying in stay-green were grown under a fully irrigated control, postflowering water deficit, and terminal (pre- and postflowering) water deficit. Water deficit reduced GLAM by 67% in the terminal drought treatment compared with the fully irrigated control. Under terminal water deficit, hybrids possessing the B35 and KS19 sources of stay-green retained more GLAM (1260 cm(2) plant(-1)) compared with intermediate (780 cm(2) plant(-1)) and senescent (670 cm(2) plant(-1)) hybrids. RQL12 hybrids (KS19 source of stay-green) displayed delayed onset and reduced rate of senescence; A35 hybrids displayed only delayed onset. Visual rating of green leaf retention was highly correlated with measured GLAM, although this procedure is constrained by an inability to distinguish among the functional mechanisms determining the phenotype. Linking functional rather than phenotypic differences to molecular markers may improve the efficiency of selecting for traits such as stay-green.

Nitrogen dynamics and the physiological basis of stay-green in sorghum

Sorghum [Sorghum bicolor (L,) Moench] hybrids containing the stay-green trait retain more photosynthetically active leaves under drought than do hybrids that do not contain this trait. Since the Longevity and photosynthetic capacity of a leaf are related to its N status, it is important to clarify the role of N in extending leaf greenness in stay-green hybrids. Field studies were conducted in northeastern Australia to examine the effect of three water regimes and nine hybrids on N uptake and partitioning among organs. Nine hybrids varying in the B35 and KS19 sources of stay-green were grown under a fully irrigated control, post-flowering water deficit, and terminal water deficit. For hybrids grown under terminal water deficit, stay-green was viewed as a consequence of the balance between N demand by the grain and N supply during gain filling. On the demand side, grain numbers were 16% higher in the four stay-green than in the five senescent hybrids. On the supply side, age-related senescence provided an average of 34 and 42 kg N ha(-1) for stay-green and senescent hybrids, respectively. In addition, N uptake during grain filling averaged 116 and 82 kg ha(-1) in stay-green and senescent hybrids. Matching the N supply from these two sources with grain N demand found that the shortfall in N supply for grain filling in the stay-green and senescent hybrids averaged 32 and 41 kg N ha(-1) resulting in more accelerated leaf senescence in the senescent hybrids. Genotypic differences in delayed onset and reduced rate of leaf senescence were explained by differences in specific leaf nitrogen and N uptake during grain filling. Leaf nitrogen concentration at anthesis was correlated with onset (r = 0.751**, n = 27) and rate (r = -0.783**, n = 27) of leaf senescence ender terminal water deficit.

Cryptococcus nyarrowii sp nov., a basidiomycetous yeast from Antarctica

in December 1997,196 soil and snow samples were collected from Vestvold Hills, Davis Base, Antarctica. Two isolates, CBS 8804 T (pink colonies) and CBS 8805 (yellow colonies), were shown by proteome analysis and DNA sequencing to represent the same species. Results from the sequencing of the D1/D2 region of the large rDNA subunit placed this species in the hymenomycetous tree in a unique sister clade to the Trichosporonalles and the Tremellalles. The clade consists of Holtermannia corniformis CBS 6979 and CBS strains 8804(T) 8805, 8016, 7712, 7713 and 7743. Morphological and physiological characteristics placed this species in the genus Cryptococcus, with characteristics including the assimilation Of D-glucuronate and myo-inositol, no fermentation, positive Diazonium blue B and urease reactions, absence of sexual reproduction and production of starch-like compounds. Fatty acid analysis identified large proportions of polyunsaturated lipids, mainly linolleic (C-18.2) and, to a lesser extent, linolenic (C-18.3) acids. On the basis of the physiological and phylogenetic data, isolates CBS 8804(T) and CBS 8805 are described as Cryptococcus nyarrowii sp. nov.

Effect of storage time and conditions on the hardness and cooking quality of adzuki (Vigna angularis)

A storage trial of two varieties of adzuki (Vigna angularis), Bloodwood and Erimo, produced in Australia, was conducted to determine the effect of various combinations of temperature, humidity and length of storage on bean quality. The beans were stored for up to 6 mo under the following conditions: temperature (10, 20 and 30degreesC), relative humidity (RH) (40 and 65%). Storage of adzuki at elevated temperature (30degreesC) and low relative humidity (40%) resulted in the greatest loss of bean moisture, increase in hydration times and decrease in bean cooking quality, i.e. increased hardness of cooked beans. The best storage conditions for the preservation of adzuki quality were 10degreesC and 65% RH.

Incorporation of bovine dry blood plasma into biscuit flour for the production of pasta

Spray-dried blood plasma (DBP) (10.9 g/100 g [w/w] nitrogen) was added to medium-protein biscuit flour (1.4 g/100 g N) during pasta manufacture. High-protein durum semolina (2.0 g/100 g N) Was used to produce the control pasta. Sensory data indicated that the addition of DBP produced pasta with significantly better colour intensity and acceptability. aroma intensity, flaN our intensity. textural strength, texture acceptability, aftertaste intensity, aftertaste acceptability. and overall acceptability The DBP/biscuit flour formulation that gave the optimum balance between pasta protein content and organoleptic acceptability contained 2.2 g/100 g DBP. A higher content of DBP resulted in increased protein levels, but these pasta formulations, ere less acceptable organoleptically. (C) 2002 Swiss Society of Food Science and Technology. Published by Elsevier Science Ltd. All rights reserved.

Evidence of thermostable amylolytic activity from Rhizopus microsporus var. rhizopodiformis using wheat bran and corncob as alternative carbon source

Rhizopus microsporus var. rhizopodiformis produced high levels of alpha-amylase and glucoamylase under solid state fermentation, with several agricultural residues, such as wheat bran, cassava flour, sugar cane bagasse, rice straw, corncob and crushed corncob as carbon sources. These materials were humidified with distilled water, tap water, or saline solutions-Segato Rizzatti (SR), Khanna or Vogel. The best substrate for amylase production was wheat bran with SR saline solution (1:2 v/v). Amylolytic activity was still improved (14.3%) with a mixture of wheat bran, corncob, starch and SR saline solution (1:1:0.3:4.6 w/w/w/v). The optimized culture conditions were initial pH 5, at 45 degrees C during 6 days and relative humidity around 76%. The crude extract exhibited temperature and pH optima around 65 degrees C and 4-5, respectively. Amylase activity was fully stable for 1 h at temperatures up to 75 degrees C, and at pH values between 2.5 and 7.5.

Purification and biochemical characterization of a thermostable extracellular glucoamylase produced by the thermotolerant fungus Paecilomyces variotii

An extracellular glucoamylase produced by Paecilomyces variotii was purified using DEAE-cellulose ion exchange chromatography and Sephadex G-100 gel filtration. The purified protein migrated as a single band in 7% PAGE and 8% SDS-PAGE. The estimated molecular mass was 86.5 kDa (SDS-PAGE). Optima of temperature and pH were 55 degrees C and 5.0, respectively. In the absence of substrate the purified glucoamylase was stable for 1 h at 50 and 55 degrees C, with a t(50) of 45 min at 60 degrees C. The substrate contributed to protect the enzyme against thermal denaturation. The enzyme was mainly activated by manganese metal ions. The glucoamylase produced by P. variotii preferentially hydrolyzed amylopectin, glycogen and starch, and to a lesser extent malto-oligossacarides and amylose. Sucrose, p-nitrophenyl alpha-D-maltoside, methyl-alpha-D-glucopyranoside, pullulan, alpha- and beta-cyclodextrin, and trehalose were not hydrolyzed. After 24 h, the products of starch hydrolysis, analyzed by thin layer chromatography, showed only glucose. The circular dichroism spectrum showed a protein rich in alpha-helix. The sequence of amino acids of the purified enzyme VVTDSFR appears similar to glucoamylases purified from Talaromyces emersonii and with the precursor of the glucoamylase from Aspergillus oryzae. These results suggested the character of the enzyme studied as a glucoamylase (1,4-alpha-D-glucan glucohydrolase).

Purification and characterization of a thermostable alpha-amylase produced by the fungus Paecilomyces variotii

An alpha-amylase produced by Paecilomyces variotii was purified by DEAE-cellulose ion exchange chromatography, followed by Sephadex G-100 gel filtration and electroelution. The alpha-amylase showed a molecular mass of 75 kDa (SDS-PAGE) and pl value of 4.5. Temperature and pH optima were 60 degrees C and 4.0, respectively. The enzyme was stable for 1 h at 55 degrees C, showing a t(50) of 53 min at 60 degrees C. Starch protected the enzyme against thermal inactivation. The a-amylase was more stable in alkaline pH. It was activated mainly by calcium and cobalt, and it presented as a glycoprotein with 23% carbohydrate content. The enzyme preferentially hydrolyzed starch and, to a lower extent, amylose and amylopectin. The K(m) of alpha-amylase on Reagen (R) and Sigma (R) starches were 4.3 and 6.2 mg/mL, respectively. The products of starch hydrolysis analyzed by TLC were oligosaccharides such as maltose and maltotriose. The partial amino acid sequence of the enzyme presented similarity to alpha-amylases from Bacillus sp. These results confirmed that the studied enzyme was an a-amylase ((1 -> 4)-alpha-glucan glucanohydrolase). (C) 2010 Elsevier Ltd. All rights reserved.

Properties of a purified thermostable glucoamylase from Aspergillus niveus

A glucoamylase from Aspergillus niveus was produced by submerged fermentation in Khanna medium, initial pH 6.5 for 72 h, at 40A degrees C. The enzyme was purified by DEAE-Fractogel and Concanavalin A-Sepharose chromatography. The enzyme showed 11% carbohydrate content, an isoelectric point of 3.8 and a molecular mass of 77 and 76 kDa estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis or Bio-Sil-Sec-400 gel filtration, respectively. The pH optimum was 5.0-5.5, and the enzyme remained stable for at least 2 h in the pH range of 4.0-9.5. The temperature optimum was 65A degrees C and retained 100% activity after 240 min at 60A degrees C. The glucoamylase remained completely active in the presence of 10% methanol and acetone. After 120 min hydrolysis of starch, glucose was the unique product formed, confirming that the enzyme was a glucoamylase (1,4-alpha-d-glucan glucohydrolase). The K (m) was calculated as 0.32 mg ml(-1). Circular dichroism spectroscopy estimated a secondary structure content of 33% alpha-helix, 17% beta-sheet and 50% random structure, which is similar to that observed in the crystal structures of glucoamylases from other Aspergillus species. The tryptic peptide sequence analysis showed similarity with glucoamylases from A. niger, A. kawachi, A. ficcum, A. terreus, A. awamori and A. shirousami. We conclude that the reported properties, such as solvent, pH and temperature stabilities, make A. niveus glucoamylase a potentially attractive enzyme for biotechnological applications.

Purification and biochemical characterization of a novel alpha-glucosidase from Aspergillus niveus

An extracellular alpha-glucosidase produced by Aspergillus niveus was purified using DEAE-Fractogel ion-exchange chromatography and Sephacryl S-200 gel filtration. The purified protein migrated as a single band in 5% PAGE and 10% SDS-PAGE. The enzyme presented 29% of glycosylation, an isoelectric point of 6.8 and a molecular weight of 56 and 52 kDa as estimated by SDS-PAGE and Bio-Sil-Sec-400 gel filtration column, respectively. The enzyme showed typical alpha-glucosidase activity, hydrolyzing p-nitrophenyl alpha-d-glucopyranoside and presented an optimum temperature and pH of 65A degrees C and 6.0, respectively. In the absence of substrate the purified alpha-glucosidase was stable for 60 min at 60A degrees C, presenting t (50) of 90 min at 65A degrees C. Hydrolysis of polysaccharide substrates by alpha-glucosidase decreased in the order of glycogen, amylose, starch and amylopectin. Among malto-oligosaccharides the enzyme preferentially hydrolyzed malto-oligosaccharide (G10), maltopentaose, maltotetraose, maltotriose and maltose. Isomaltose, trehalose and beta-ciclodextrin were poor substrates, and sucrose and alpha-ciclodextrin were not hydrolyzed. After 2 h incubation, the products of starch hydrolysis measured by HPLC and thin layer chromatography showed only glucose. Mass spectrometry of tryptic peptides revealed peptide sequences similar to glucan 1,4-alpha-glucosidases from Aspergillus fumigatus, and Hypocrea jecorina. Analysis of the circular dichroism spectrum predicted an alpha-helical content of 31% and a beta-sheet content of 16%, which is in agreement with values derived from analysis of the crystal structure of the H. jecorina enzyme.