Isolation and thermal characterization of an acidic isoperoxidase from turnip roots

An acidic peroxidase (pI approximately 2.5) was purified from turnip roots (TAP), and its thermal properties were evaluated. TAP is a monomeric protein having a molecular weight (MW) of 49 kDa and a carbohydrate content accounting for 18% of the MW. The yield of pure TAP was relatively high ( approximately 2 mg/kg of fresh roots), with a specific activity of 1810 2,2'-azinobis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) units/mg at pH 6. The activity increased 4-fold at the optimum pH (4.0) to 7250 ABTS units/mg, higher than that of most peroxidases. TAP was heat stable; heat treatment of 25 min at 60 degrees C resulted in 90% initial activity retention, whereas an activity of 20% was retained after 25 min of heating at 80 degrees C. TAP regained 85% of its original activity within 90 min of incubation at 25 degrees C, following heat treatment at 70 degrees C for 25 min. Thermal inactivation caused noticeable changes in the heme environment as evaluated by circular dichroism and visible spectrophotometry. TAP was rapidly denatured by heating in the presence of 1.0 mM ethylene glycol bis(beta-aminoethyl ether) N,N,N',N'-tetraacetic acid, but the Soret band and activity were fully recovered by adding an excess of Ca(2+). This is further evidence that Ca(2+) plays an important role in the stability of TAP. The high specific activity of TAP, together with its relatively high thermal stability, has high potential for applications in which a thermally stable enzyme is required.     

Genetic structure of Xiphinema pachtaicum and X. index populations based on mitochondrial DNA variation

The dagger nematodes Xiphinema pachtaicum and X. index are two of the most widespread and frequently occurring Xiphinema spp. co-infesting vineyards and other crops and natural habitats worldwide. Sexual reproduction is rare in these species. The primary objective of this study was to determine the genetic structure of X. pachtaicum and X. index populations using eight and seven populations, respectively, from different "wine of denomination of origin (D.O.) zones" in Spain and Sardinia (Italy), by studying mitochondrial (cytochrome oxidase c subunit 1 or COI) and nuclear (D2-D3 expansion segments of 28S rDNA) markers. Both Xiphinema spp. showed low intraspecific divergence among COI sequences, ranging from 0.2% (1 base substitution) to 2.3% (10 substitutions) in X. pachtaicum and from 0.2% (1 base substitution) to 0.4% (2 substitutions) in X. index. Population genetic structure was strong for both species. Nevertheless, molecular differences among grapevine-growing areas were not significant, and intrapopulation diversity was very low. It is hypothesized that this genetic homogeneity in the nematode populations reflects their predominant parthenogenetic reproduction mode and low dispersal abilities. Our results also show that X. pachtaicum populations in Spain have possibly been established from two different populations of origin. Results also demonstrated that the two DNA regions studied are suitable diagnostic markers for X. index and X. pachtaicum.     

In planta and soil quantification of Fusarium oxysporum f. sp. ciceris and evaluation of Fusarium wilt resistance in chickpea with a newly developed quantitative polymerase chain reaction assay

Fusarium wilt of chickpea caused by Fusarium oxysporum f. sp. ciceris can be managed by risk assessment and use of resistant cultivars. A reliable method for the detection and quantification of F. oxysporum f. sp. ciceris in soil and chickpea tissues would contribute much to implementation of those disease management strategies. In this study, we developed a real-time quantitative polymerase chain reaction (q-PCR) protocol that allows quantifying F. oxysporum f. sp. ciceris DNA down to 1 pg in soil, as well as in the plant root and stem. Use of the q-PCR protocol allowed quantifying as low as 45 colony forming units of F. oxysporum f. sp. ciceris per gram of dry soil from a field plot infested with several races of the pathogen. Moreover, the q-PCR protocol clearly differentiated susceptible from resistant chickpea reactions to the pathogen at 15 days after sowing in artificially infested soil, as well as the degree of virulence between two F. oxysporum f. sp. ciceris races. Also, the protocol detected early asymptomatic root infections and distinguished significant differences in the level of resistance of 12 chickpea cultivars that grew in that same field plot infested with several races of the pathogen. Use of this protocol for fast, reliable, and cost-effective quantification of F. oxysporum f. sp. ciceris in asymptomatic chickpea tissues at early stages of the infection process can be of great value for chickpea breeders and for epidemiological studies in growth chambers, greenhouses and field-scale plots.     

Evaluation of factors affecting vaccine efficacy of recombinant Marek's disease virus lacking the Meq oncogene in chickens

We previously reported that deletion of the Meq gene from the oncogenic rMd5 virus rendered it apathogenic for chickens. Here we examined multiple factors affecting Marek's disease vaccine efficacy of this nonpathogenic recombinant Meq null rMd5 virus (rMd5deltaMeq). These factors included host genetics (MHC haplotype), strain or dose of challenge virus, vaccine challenge intervals, and maternal antibody status of the vaccinated chicks. Studies on host genetics were carried out in five chicken lines comprising four different MHC B-haplotypes. Results showed that chicken lines tested were highly protected, with protective indexes of 100% (B*2/*15), 94% (B*2/*2), 87% (B*19/*19), and 83% (B*21/*21). At a challenge dose above 8000 plaque-forming units, differences in protection were observed between the two highly virulent strains examined (648A and 686). The interval between vaccination and challenge indicated a protective efficacy from 0 to 2 days varied greatly (12%-82%) after challenge with vv+686, the most virulent virus. Less variation and significant protection began at 3 days post vaccination and reached a maximum at 5 days post vaccination with about 80%-100% protection. Taken together, our results indicate that the factors examined in this study are important for vaccine efficacy and need to be considered in comparative evaluations of vaccines.     

Performance of new and commercial xylanases for ECF and TCF bleaching of eucalyptus kraft pulp

Since xylanases can differ widely in their bleaching efficiency, the performance of one new and two commercial xylanases was evaluated in an eucalyptus kraft pulp following XD (X: xylanase; D: chlorine dioxide) and XP (P: hydrogen peroxide) sequences. The new xylanase did not show a significant bleach boosting effect but increased the hexenuronic acid (HexA) removal by 10% after the D stage. The two commercial xylanases behaved in a different way, being one of them (X_C) the most effective in increasing delignification (9%) and brightness (3%ISO). Its effectiveness was related to its greater action on releasing the xylan polymer, thus producing also a strong decrease in the HexA contents during the enzymatic stage (15%). All xylanases produced morphological changes in the fibre surfaces, but only with X_C cracks and holes that improved the diffusion of reactives were observed. Finally, the best bleaching results were obtained with the XD sequence and therefore, a complete bleaching sequence XDEopD₁ (Eop: alkaline extraction with oxygen and peroxide) was carried out with the best enzyme.     

First report of a phytoplasma associated with Spondias purpurea (Jocote de Corona) in El Salvador

A new disease of the fruit tree Jocote de Corona (Spondias purpurea L.) was observed in El Salvador, Central America. The symptoms included small chlorotic leaves, highly proliferating shoots, and shortened internodes. Absence of sweet pulp in fruits made them inedible, causing considerable yield losses for farmers. The disease etiology was investigated using polymerase chain reaction with phytoplasma-specific primers, sequencing, and phylogenetic analysis. We obtained no amplification products from symptomless plants, whereas all tests were positive from plants with symptoms. Phylogenetic analysis indicated that this phytoplasma clustered in the 16S rIII group, the type member of which is X-disease phytoplasma. This is the first report of a phytoplasma associated with Jocote de Corona disease in El Salvador and Central America. The nucleotide sequence data reported are available in the DDBJ/EMBL/GenBank databases under the accession number AJ888471     

Charcoal production at kiln sites affects C and N dynamics and associated soil microorganisms in Quercus spp. temperate forests of central Mexico

Temperate forests dominated by Quercus spp. cover large parts of Central Mexico and rural communities depend on these forests for wood and charcoal. The impacts of charcoal production on selected chemical properties including C and N dynamics, and populations of ammonifiers, nitrifiers and denitrifiers were investigated on surface soils (0–15 cm) collected during the dry and rainy season of these forests. Organic C was halved in soil at the kiln sites compared to undisturbed forest soil. Concentrations of exchangeable Ca²⁺, K⁺ and Mg²⁺ increased >1.6 times at kiln sites and pH increased from 4.5 in undisturbed soil to 7.0 at kiln sites. The kiln sites had 1.3 times and 2.4 times lower microbial biomass C and N, respectively, than undisturbed forest sites during the rainy season. Although the effect of charcoal production on NH₄⁺, NO₂^? and NO₃^? concentrations was small, the ammonifying, nitrifying and denitrifiers were 16 times lower at the kiln sites than in the undisturbed forest soil. This research found that the charcoal production had a negative effect on the cultivable microorganisms involved in N cycling and the soil microbial biomass C and N compared to undisturbed forest soil. Differences in inorganic N dynamics were more affected by seasonality, i.e. precipitation, than by charcoal production.     

Carbohydrate composition of sugarcane cultivars that are resistant or susceptible to <Emphasis Type="Italic">Sugarcane yellow leaf virus</Emphasis>

Sugarcane cultivars with a high (susceptible cultivars) and low (resistant cultivars) virus titer of Sugarcane yellow leaf virus were grown in the field. The carbohydrate composition in green leaf tops and in stems was determined. In RT-PCR of leaf extracts, susceptible cultivars had a high SCYLV-titer, whereas resistant cultivars had a very low titer. The cultivars differed in biomass yield, but these differences were not correlated with susceptibility. However, carbohydrate composition did have susceptibility-specific differences. Hexose levels were lower in green leaf tops and stalks of susceptible (strongly infected) cultivars than in those of resistant (weakly infected) cultivars. The stalks of susceptible cultivars also had less starch than those of resistant cultivars. Thus, the viral susceptibility (and infection) affected sugar metabolism. In addition, a positive correlation between hexose and starch in stems and between hexose and sucrose in green leaf tops was observed. The results from susceptible versus resistant cultivars were the opposite of those in the comparison between infected versus virus-free lines of the same cultivar. The breeding process apparently had unintentionally selected clones with modulated carbohydrate metabolism to avoid or compensate for the adverse effects of SCYLV infection.