Proteomic analysis reveals suppression of bark chitinases and proteinase inhibitors in citrus plants affected by the citrus sudden death disease

Citrus sudden death (CSD) is a disease of unknown etiology that greatly affects sweet oranges grafted on Rangpur lime rootstock, the most important rootstock in Brazilian citriculture. We performed a proteomic analysis to generate information related to this plant pathogen interaction. Protein profiles from healthy, CSD-affected and CSD-tolerant stem barks, were generated using two-dimensional gel electrophoresis. The protein spots were well distributed over a pI range of 3.26 to 9.97 and a molecular weight (MW) range from 7.1 to 120 kDa. The patterns of expressed proteins on 2-DE gels made it possible to distinguish healthy barks from CSD-affected barks. Protein spots with MW around 30 kDa and pI values ranging from 4.5 to 5.2 were down-regulated in the CSD-affected root-stock bark. This set of protein spots was identified as chitinases. Another set of proteins, ranging in pI from 6.1 to 9.6 with an MW of about 20 kDa, were also suppressed in CSD-affected rootstock bark; these were identified as miraculin-like proteins, potential trypsin inhibitors. Down-regulation of chitinases and proteinase inhibitors in CSD-affected plants is relevant since chitinases are well-known pathogenesis-related protein, and their activity against plant pathogens is largely accepted.     

Management of strawberry anthracnose by choice of irrigation system,mulching material and host resistance

We studied, by means of field experiments, the combined effects of irrigation system, mulching material and genetic resistance on the incidence of strawberry anthracnose caused by Colletotrichum acutatum. All trials involved artificial inoculation with fruits colonized by the pathogen. One experiment examined the effects of drip irrigation, overhead sprinkler irrigation, and localized low-pressure tape irrigation, and the effects of grass (Brachiaria sp.) and plastic (polyethylene) mulches, in a split plot, randomized complete block design with four replicates. Lower disease incidence, indicating lower pathogen dispersal, was verified with low-pressure tape and drip irrigation. Flower blight and fruit rot incidence in drip-irrigated plots was minimal (ca. 2% of the disease levels in the plots otherwise irrigated). Grass mulch reduced flower blight in the sprinkler system (up to 80%), but had no significant effect when disease levels were already low due to use of other irrigation systems. A second experiment compared the effects of grass mulch, pine (Pinus elliotti) mulch, and plastic mulch with overhead sprinkler irrigation on strawberry cultivars partially resistant (‘Dover’) and susceptible (‘Campinas’) to the disease in a factorial completely randomized design (CRD). Both organic mulches reduced flower blight and diseased fruit incidence by up to 76% compared to the plastic mulch treatment, especially in the early stages of the epidemics. ‘Dover’ had 70% lower incidence of flower blight than ‘Campinas’, and the effects of organic mulches were more pronounced in ‘Campinas’. A third experiment, with all three irrigation systems combined with inoculated and non-inoculated plots, estimated yield effects due to disease and irrigation system. All plots were covered with plastic mulch in a CRD with six treatments. This experiment clearly confirmed the higher flower blight and fruit rot incidences when sprinkler irrigation was used. In inoculated treatments, fruit yield was significantly reduced in tape-irrigated plots, and more so in sprinkler-irrigated plots. In drip-irrigated plots yield was reduced by only 1%. Overall, our results confirm the importance of cultural and genetic factors as valuable means of strawberry anthracnose management. Adoption of localized irrigation systems, the use of organic mulches and choice of a partially resistant cultivar significantly reduced disease levels in field plots. The importance of water splash for C. acutatum dispersal and its dependency on the soil mulch characteristics are the likely causes for the results observed.     

Supercritical fluid extraction of 5-hydroxymethyl-2-furaldehyde from raisins

An extraction method based on supercritical CO(2) has been developed for the analysis of 5-hydroxymethyl-2-furaldehyde in raisins. To optimize extraction variables, a fractional factorial experimental design was applied. Six extraction variables were optimized. The organic modifier used for increasing the extraction fluid solvating power was the most important factor. Methanol as organic modifier produced 10-fold higher recoveries of 5-hydroxymethyl-2-furaldehyde than ethyl acetate. The efficiency of the organic modifier in the static extraction phase was compared with using it in the dynamic extraction phase. Repeatability of the analysis method was evaluated, which resulted in an RSD of <5%. 5-Hydroxymethyl-2-furaldehyde was quantified in raisins, and the concentration was found to be 0.128 mg/g of raisin.     

Evaluation of spatial and temporal variability of pasture based on topography and the quality of the rainy season

Yield and botanical composition of a given dry-land pasture are heavily influenced by rainfall distribution, and vary according to topography. Through analyzing these parameters, it is possible to identify patterns of spatial distribution, related to topographic characteristics, which can be used to improve spatial management of pasture. The main objective of this project was to assess the role of rainfall, temperature and topography in the spatial and temporal variability of pasture and establish models for predicting the spatial distribution and yield of certain plant species, based on its topographic characteristics. This study was carried out over three years, 2004 to 2006, in a dry-land pasture located in Southern Portugal. The data obtained were analyzed as a function of distance to flow lines and the results demonstrate that certain topographical characteristics, associated with agronomic information, can be very useful in explaining the spatial and temporal variability of yield and the botanical composition of the pasture. The slope of the linear regression of the variables mentioned can be used to estimate the spatial variation of yield and the botanical composition as a function of distance to flow lines. The regression coefficient can be estimated from the annual rainfall, given the strong correlation between the two.     

Chemical and microbiological parameters for the characterisation of the stability and maturity of pruning waste compost

Composting of pruning waste, leaves and grass clippings was monitored by different parameters. A windrow composting pile, having the dimensions 2.5 m (height) x 30 m (length) was establish. The maturation of pruning waste compost was accompanied by a decline in NH₄ ⁺-N concentration, water soluble C (WSC) and an increase in NO₃ ^–-N content. Both organic matter (OM) content and total N (TN) losses during composting followed a first-order kinetic equation. These results were in agreement with the microbiological activity measured either by the CO₂ respiration or dehydrogenase (DH-ase) activity during the process. Statistically significant correlations were found between DH-ase activity, easily biodegradable organic C forms, NH₄ ⁺-N and NO₃ ^–-N concentrations and organic matter content and N losses. For this reason, DH-ase activity and the CO₂ evolution could be used as good indicators of pruning waste compost maturity. In contrast, humification parameters data from the organic matter fractionation did not agree with the initially expected values and did not contribute to the assessment of compost maturity. Neither the cation exchange capacity nor the germination index showed a clear tendency during the composting time, suggesting that these parameters are not suitable for evaluating the dynamics of the process.     

Influence of tillage system on denitrification in maize-cropped soils

Denitrification losses show an irregular pattern through the year, often being caused by climatic conditions and management practices. The objectives of the present work were to quantify denitrification losses and to determine the influence of tillage system on the factors that control denitrification in fertilized soils. The modal profile of the soil was an Vertic Argiudoll, clay loam texture, located in Buenos Aires province, Argentina. The treatments were: (a) fertilized, (b) incorporated fertilization and (c) without fertilization for both no tillage and conventional tillage systems. Chambers were placed in the field to measure denitrification. In this clayish soil the estimated mean values of accumulated denitrification during the crop cycle (90 days) were 0.190kgNha^–1 for conventional tillage and 0.350kgNha^–1 for no tillage. In treatments with no tillage, losses by denitrification were approximately twice those of conventional tillage. These differences were also evidenced by the number of microorganisms, which were significantly higher (P<>;5%) for no tillage on all dates, except for at flowering. The increase at flowering coincided with the period of highest rainfall and consequently the highest water contents in the soil. The highest denitrification losses, except for sowing, were measured when soil moisture content was more than 30% (v/v). Denitrification increased in conjunction with an increase in the availability of carbon that is consumed by the heterotrophic microorganisms (including the denitrifiers). Received: 30 July 1996     

Functional Morphology of the Zona Pellucida

The zona pellucida (ZP) is an extracellular matrix surrounding the oocyte and the early embryo that exerts several important functions during fertilization and early embryonic development. The ZP of most mammalian species is composed of three major glycoproteins that show considerable heterogeneity due to extensive post-translational modifications. Sodium dodecyl sulphate polyacrylamide gel electrophoresis of the ZP reveals three to four glycoproteins which have been nominated ZPI. ZP2, ZP3 and ZP4. As cloning and characterization of the ZP genes of a variety of mammalian species including domestic animals show a high homology, three classes of ZP genes, ZPA, ZPB and ZPC can be discerned. The corresponding proteins were named ZPA, ZPB and ZPC. Whereas in the mouse ZPB is the primary sperm receptor. the situation is more complicated in other species. For instance, in the pig ZPA has been shown to possess receptor activity. Interaction between gametes during fertilization is at least in part regulated by carbohydrate moieties of the ZP and carbohydrate-binding proteins of the sperm surface. In domestic animals zona proteins are expressed in both the oocyte and granulosa cells in a stage-specific pattern and may play a role in granulosa cell differentiation. The role of ZP glycoproteins in immunocontraception is briefly discussed.