Biometrical, biological, biochemical and molecular characteristics of Meloidogyne incognita isolates and related species

Meloidogyne incognita is one of the most polyphagous species of root-knot nematodes occurring in Brazil and worldwide. Eight M. incognita isolates were studied, representing two enzymatic phenotypes (esterase and malate desydrogenase: I1/N1, I2/N1) and four cryptic Meloidogyne sp.1 (S2/N1) isolates, representing one cytological type (3n?=?40–46). Three M. hispanica isolates (Hi3/N1, 2n?=?32–36) and two of an atypical Meloidogyne sp.2 (S2a/N3, 3n?=?40–44) were included in this study for comparison. All isolates were tested with three M. incognita-specific molecular markers. The primer pairs B06F/R, miF/R and incK14F/R amplified three species-specific fragments of 1,200?bp, 955?bp and 399?bp, respectively for M. incognita and Meloidogyne sp.1 isolates. No amplification occurred in the M. hispanica and Meloidogyne sp.2 isolates, except with primers miF/R (1,650?bp). The genetic variability of the Meloidogyne spp. isolates was evaluated, using RAPD and ISSR markers. The phylogenetic analyses revealed two strongly supported monophyletic clades: clade I, consisting of M. hispanica and the atypical Meloidogyne sp.2 isolates, and clade II, clustering together all M. incognita and the Meloidogyne sp.1 isolates. Considering the biometrical, cytological and molecular approaches, it was possible to conclude that the isolates with three enzymatic phenotypes (I1/N1, I2/N1 and S2/N1) presented the characteristics described for M. incognita. Some correlations were detected between the isozymatic phenotypes and the tree topology (S2a/N3, Hi3/N1, I1/N1, S2/N1), but no strict correlation could be observed for the phenotype I2/N1 and one isolate of S2/N1. Morphologically, the Msp.2 isolates differ from M. incognita and M. hispanica by the female stylet features presenting straight cone tip and round pear shaped knobs, posteriorly sloping. The results of this study suggested that the Msp.2 isolates with phenotypes S2aN3 belong to a new or an unidentified species closely related to M. hispanica.     

Low genetic variability in Sclerotinia sclerotiorum populations from common bean fields in Minas Gerais State,Brazil, at regional,local and micro‐scales

Sclerotinia sclerotiorum, causal agent of white mould, is the most destructive and widely distributed soilborne pathogen of common bean during the autumn–winter season in Brazil. Nevertheless, little is known about the genetic structure of the pathogen population. Microsatellite (SSR) markers and mycelial compatibility groups (MCGs) were used to characterize 118 isolates collected from 20 bean fields located in the most important growing regions of Minas Gerais State (MG). Additionally, the genetic variability among 10 isolates obtained from a single sclerotium was investigated in 10 different sclerotia. Seventy SSR haplotypes and 14 MCGs were identified among the 118 isolates. The genetic differences within bean growing areas accounted for most of the genetic variation (72%). Despite the relatively high genotypic diversity, the SSR loci were at linkage disequilibrium. Moreover, 70% of the isolates were assigned to only two MCGs, and haplotypes of a given MCG were closely related. The discriminant analysis of principal components revealed five groups. There was strong genetic differentiation between isolates collected in one municipality in southern MG when compared to other regions. Common bean resistance to white mould should be assessed with representative isolates of the five genetic groups and, if possible, of the different MCGs detected in the present study. One to five haplotypes were detected among the 10 isolates obtained from a single sclerotium. Therefore, in order to ensure genetic identity of an isolate, hyphal tip or monoascosporic isolates should be used.     

Biofilm-forming ability profiling of Staphylococcus aureus and Staphylococcus epidermidis mastitis isolates

Biofilm-forming ability has been increasingly recognized as an important virulence factor in Staphylococci, facilitating their persistence in the host, evading its defences and allowing bacterial survival at high antimicrobial concentrations. Staphylococcus aureus remains a major pathogen of chronic mastitis, but in the last years Staphylococcus epidermidis has emerged as a relevant mastitis pathogen. The present work aimed at the evaluation of the biofilm-forming ability of Staphylococci field isolates from bovine subclinical mastitis and at the development of a fluorescent in situ hybridisation (FISH) protocol that would allow the direct observation of biofilm formation in milk samples. The analysis of phenotypic expression in Congo Red Agar (CRA) and by FISH, showed that 37.5% of the S. aureus isolates produced biofilm, while by optical density measurement only 18.75% isolates revealed this phenotype. The results showed a fair agreement according to the kappa coefficient test (kappa = 0.259). Regarding S. epidermidis mastitis isolates, 37.5% revealed the ability to produce biofilm, but only four isolates were positive by all methods. This agreement was moderate (kappa = 0.467). The application of FISH to artificially contaminated milk samples allowed the direct observation of biofilm production by 37.5% isolates, showing total agreement with the CRA results. This method better mimics the in vivo conditions, especially in terms of the presence of calcium and iron, which in high concentrations, respectively, are known to inhibit or induce biofilm production.     

Mineral requirements of hair sheep in tropical climates

In this study, we evaluated the requirements of calcium (Ca), phosphorus (P), magnesium (Mg), sodium (Na) and potassium (K) for sheep hair growth. Experimental diets contained different levels of metabolizable energy [ME; 0.96, 1.28, 1.72, 2.18 and 2.62 Mcal/kg of dry matter, (DM), corresponding to 4.23, 5.64, 7.58, 9.61 and 11.55 MJ/kg DM]. The lambs' hair (n = 48) at 2 months of age presented an average body weight (BW) of 12.05 ± 1.81 kg. At the beginning of the experiment, eight animals were slaughtered as a reference group to estimate the initial empty body weight and body composition. Net mineral requirements (g/day) ranged from 0.73 to 0.71 g of Ca, 0.51 to 0.49 g of P, 0.026 to 0.026 g of Mg, 0.16 to 0.19 g of Na and 0.15 to 0.13 g of K for animals with a BW ranging from 15 to 30 kg and a daily gain of 100 g. The results of this study indicate that the net macromineral requirements for weight gain in Morada Nova lambs are different from the values commonly recommended by the Agricultural and Food Research Council.     

Genetic diversity of the root‐knot nematode Meloidogyne enterolobii and development of a SCAR marker for this guava‐damaging species

The objectives of this work were to evaluate the genetic variability of Meloidogyne enterolobii by molecular markers, and develop species‐specific molecular markers for application in detection. Sixteen M. enterolobii isolates from different geographical regions (Brazil and other countries) and hosts were used in this study. The identification and purification of the populations were carried out based on isoenzyme phenotype. The DNA amplification of the intergenic region (IGS) of the rDNA and of the region between the cytochrome oxidase subunit II (COII) and 16S rRNA genes (mtDNA) produced specific fragments of the expected size for this nematode, i.e. 780 and 705 bp, respectively. Intraspecific variability among the isolates was evaluated with three different neutral molecular markers: AFLP, ISSR and RAPD. The results showed a low level of diversity among the isolates tested, indicating that M. enterolobii is a genetically homogeneous root‐knot nematode species. The RAPD method allowed the identification of a species‐specific RAPD fragment for M. enterolobii. This fragment was cloned and sequenced, and from the sequence obtained, a set of primers was designed and tested. The amplification of a 520‐bp‐long fragment occurred only for the 16 isolates of M. enterolobii and not for the 10 other Meloidogyne species tested. In addition, positive detection was achieved in a single individual female, egg‐mass and second stage juvenile of this nematode. This SCAR species‐specific marker for M. enterolobii represents a new molecular tool to be used in the detection of this nematode from field samples and as a routine diagnostic test for quarantine devices .     

Effect of Leptin on In Vivo Goat Embryo Production

The aim of this study was to evaluate the effect of leptin administration during superovulation on in vivo goat embryo production. Ten mature does were superovulated with 133 mg follicle‐stimulating hormone (FSH) i.m. in six descending doses at 12‐h intervals. The goats received 4.8 μg/kg human recombinant leptin s.c. (leptin group, n = 5) or phosphate‐buffered saline (PBS) (control group, n = 5) with the first and second FSH doses. The does were mated and subjected to embryo collection by transcervical technique 6 days later. The total number of cells per embryo and the number of cells with fragmented DNA were assessed in selected blastocysts by combining Hoechst 33342 and terminal dUTP nick‐end labelling (TUNEL) staining. Plasma concentrations of oestradiol (E₂) and progesterone (P₄) were determined by electrochemiluminescence from the day of FSH treatment, on the day of superovulatory oestrus and on the day before embryo collection. Compared with the control group, the does that received leptin had a higher number of transferable embryos (p < 0.005), fewer embryos classified as degenerated (p < 0.001) and fewer TUNEL‐positive cells/blastocyst (p < 0.001). The number of transferable embryos was positively correlated with E₂ concentrations on day of oestrus (r = 0.562; p < 0.01) and P₄ concentrations on the day of embryo collection (r = 0.912; p < 0.001). We concluded that in vivo leptin administration during FSH treatment improved embryo quality and affected ovarian steroidogenesis in superovulated goats.     

Species-specific DNA markers for identification of two root-knot nematodes of coffee: Meloidogyne arabicida and M. izalcoensis

Seven root-knot nematodes (RKN), including Meloidogyne exigua, M. incognita, M. paranaensis, M. enterolobii, M. arabicida, M. izalcoensis and M. arenaria are major pathogens of coffee crop in the Americas. Species-specific primers for their identification have been developed for five of them and constitute a fast and reliable method of identification. Here we report a PCR-based assay for specific detection of M. arabicida and M. izalcoensis. Random Amplified Polymorphic DNA fragments specific for these two species were converted into sequence characterized amplified region (SCAR) markers. PCR amplification using the SCAR primers produced a specific fragment of 300 bp and 670 bp for M. arabicida and M. izalcoensis, respectively, which were absent in other coffee-associated Meloidogyne spp. tested. SCAR primers also allowed successful amplification of DNA from single second-stage juveniles (J2), males and females. In addition, these primers were able to unambiguously detect the target species in nematode suspensions extracted from soil and roots samples, in different isolates of the same species or when used in multiplex PCR reactions containing mixtures of species. These results demonstrated the effectiveness of these SCAR markers and their multiplex use with those previously developed for M. exigua, M. incognita, M. paranaensis, M. enterolobii and M. arenaria constitute an essential detection tool. This diagnostic kit will contribute for specific J2 identification of the major RKN infecting coffee from field samples in the Americas.     

Genetic-diversity assessed by microsatellites in tropical maize populations submitted to a high-intensity reciprocal recurrent selection

BR-105 and BR-106 are important tropical maize populations, which were submitted to a high-intensity reciprocal recurrent selection, generating the IG-3 and IG-4synthetics. Using 30-microsatellite loci,we measured and compared the genetic diversity of these populations and their synthetics. The populations did not differ significantly regarding the amount of genetic diversity. As a consequence of selection, genetic variability losses, in terms of mean number of alleles per locus,proportion of polymorphic loci, and gene diversity did occur and were greater in the synthetic IG-3 than IG-4. In the synthetics, the number of loci in adherence to Hardy-Weinberg proportions was superior to that observed in the populations. The Wright's mean fixation index was higher than the mean value expected for outcrossing species (5%) indicating as light excess of homozygotic individuals in both populations. The genetic distances confirmed the favourable effects of one cycle of recurrent selection, as the synthetics became more isolated in comparison to the original populations. This revised version was published online in July 2006 with corrections to the Cover Date.