Characterization of aflatoxigenic and non-aflatoxigenic strains of <Emphasis Type="Italic">Aspergillus</Emphasis> section <Emphasis Type="Italic">Flavi</Emphasis> isolated from corn grains of different geographic origins in Brazil

Aflatoxins can cause great economic losses and serious risks to humans and animals health. The largest aflatoxin producers belong to Aspergillus section Flavi and can occur naturally in food commodities. Studies showed that molecular tools as well as the type of sclerotia produced by the strains could be helpful for identification of Aspergillus species and could be correlated with levels of toxin production. The purpose of this work was to characterize the genetic diversity using AFLP technique, the type of sclerotia and the ability of aflatoxin production by isolated strains from corn of different origins in Brazil, and to verify whether qPCR based on aflR and aflP genes is appropriate for estimating the level of aflatoxin production. All the 75 strains were classified as A. flavus and the AFLP technique showed a wide intraspecific variability within them. Regarding sclerotia production, 34% were classified as S and 66% as L type. Among the aflatoxin-producers, 52.8% produced aflatoxin B₁, while 47.2% aflatoxins B₁ and B₂. Statistical analysis showed no correlation between sclerotia production and aflatoxigenicty, and no correlation between the phylogenetic clusters and aflatoxin production. Concerning the relative expression of aflR and aflP, Pearson’s correlation test demonstrated low positive correlation between the expression of the aflR and aflP genes and the production of AFB₁ and AFB₂, but showed high positive correlation between aflR and aflP expression. In contrast to the other reference strains, A. oryzae ATCC 7282 showed no amplification of aflR and aflP. The results highlight the need for detection of reliable and reproducible markers with a high positive correlation with aflatoxin production.     

Essential oils for preventative treatment and control of Asian soybean rust

The objective of the work was to evaluate the effect of essential oils as a preventive treatment to control Asian soybean rust. Initially the fungitoxic effect of the essential oils of Hyptis marrubioides, Aloysia gratissima and Cordia verbenacea was tested on the urediniospores of Phakopsora pachyrhizi, through in vitro tests. The in vivo test was set up in a greenhouse, using cultivar MGBR-46. The treatments consisted of the three oils at different concentrations, a fungicide based on pyraclostrobin + epoxyconazole and a control. To verify the potential of the essential oils in preventive treatment, inoculation was conducted at 0, 6, 12 and 24?h intervals, after application of the treatments. All treatments inhibited 100% of Phakopsora pachyrhizi germination. In the in vivo test, it was observed that all of the oils were efficient in controlling soybean rust, by preventive treatment, mainly at the higher concentrations. Efficiency of the oils was reduced with the increase of the interval between the application of the treatments and the onset of the pathogen. The essential oils, at the tested dosages, were not as efficient as the pyraclostrobin + epoxyconazole based fungicide.     

Comparing p20’s RNA silencing suppressing activity among five phylogenetic groups of <Emphasis Type="Italic">Citrus Tristeza virus</Emphasis>

The p20 protein encoded by the Citrus Tristeza Virus (CTV) was previously identified as a RNA silencing suppressor. In this study, we analyzed the p20’s suppressing activity from five phylogenetic groups of CTV, using the co-infiltration assay of Green fluorescence protein (GFP) gene and the suppressor gene in 16C line Nicotiana benthamiana plants. Green fluorescence, GFP mRNA relative levels and GFP specific siRNAS were compared showing in most cases, only slight differences. Contrary to previous studies, the p20 suppressor was not able to impede neither short range nor systemic spreading of RNA silencing. The suppressor from the phylogenetic group 4 revealed a much reduced activity when compared with the others. At present we still don’t know whether this property is a characteristic of this group or an atypical feature due to a unique point mutation. The differences in the symptom type and intensity originated by isolates belonging to the phylogenetic groups assayed could not be related to differences to the p20 suppressor’s activity.     

Coffee resistance to Colletotrichum kahawae is associated with lignification, accumulation of phenols and cell death at infection sites

Histological and ultrastructural studies were undertaken to compare Colletotrichum kahawae growth and the sequence of responses it induced in resistant and susceptible coffee genotypes. Coffee resistance was characterized by a restricted fungal growth associated with hypersensitive-like cell death and early accumulation of phenolic compounds, such as flavonoids (cytoplasmic contents) and hydroxycinnamic acid derivatives (cell walls). This accumulation of phenols in the cell walls preceded their lignification and thickening. In the susceptible genotype, a late accumulation of hydroxycinnamic acid derivatives in a number of cell walls and the encasement of some intracellular hyphae were also observed, but these delayed host responses did not prevent fungal growth and sporulation.     

Alteration of physiological parameters of Austrocedrus chilensis by the pathogen Phytophthora austrocedrae

The effect of the pathogen Phytophthora austrocedrae on tree physiology of Austrocedrus chilensis in Patagonia was studied in a 4‐week study. In the first week, stem‐inoculated saplings showed a significant decrease in photosynthesis (A) without alteration of stomatal conductance (g_s) or stem‐specific hydraulic conductivity (Ks). From the second week on, progressive decreases in A, g_s and Ks were observed, concomitantly with development of significant stem lesions. Water use efficiency (WUE) increased in the second week and declined progressively from the third week. Hyphae and resinous materials were observed in tracheids and rays below lesions. Necrosis of parenchyma ray cells and blockage of tracheids torus were observed. Healthy xylem showed no resinous materials or tracheid blockage, but abundant starch in rays, which was absent in altered xylem. The culture filtrate (CF) of the pathogen was shown to induce changes in extracellular pH and conductivity, and increased necrosis in tissues of leaves and stem challenged with CF in vitro. Similar results were obtained in leaf tissues of the inoculated saplings in vivo. CF injection into xylem of saplings induced a decline in A and disturbance of leaf tissue integrity, without altering g_s, WUE or Ks. The decrease of A correlated with changes in tissue integrity. A possible mechanism of A. chilensis decline induced by P. austrocedrae is discussed.     

Conventional and Doppler ultrasound for the differentiation of benign and malignant canine mammary tumours

Objectives : The aim of this study was to evaluate the sensitivity of conventional and Doppler ultrasound for differentiation of benign and malignant mammary tumours in female dogs. Methods : Mammary tumours were evaluated from 60 animals and divided into two distinct groups, group 1 (benign tumours) and group 2 (malignant tumours). The tumours were assessed by conventional ultrasound, Doppler ultrasound mode, histopathology and immunohistochemical detection of vascular endothelial growth factor. Results : Conventional ultrasound examination was found to be ineffective in separating tumours into the two experimental groups. Similarly, using colour‐flow Doppler ultrasound, no correlation was found between the presence of vascularisation and its characteristics between the two groups. Triplex Doppler ultrasound yielded average maximum velocities of 28·71 cm/s for malignant and 19·91 cm/s for benign tumours, which were significantly different (P=0·01). For vascular endothelial growth factor, an average score of 2·22 was found for group 2 and 1·66 for group 1 (P=0·03). Positive correlations were found between vascular endothelial growth factor and presence of vascularisation (P=0·04 and r=0·3658) and between vascular endothelial growth factor and maximum velocity (P=0·03 and r=0·3913). Clinical Significance : Doppler evaluation may be used to predict malignancy of mammary tumours in bitches.     

Inheritance of resistance to a Bacillus thuringiensis toxin in a field population of diamondback moth (Plutella xylostella)

Inheritance of resistance to the Bacillus thuringiensis Berl. CryIA(b) crystal protein was studied in Plutella xylostella L. (diamondback moth). A field population 50-fold more resistant to CryIA(b) than a control susceptible strain was used. Dose-mortality curves of the resistant population, the susceptible strain and the F₁ from the two reciprocal crosses were compared. Resistance transmission to the F₁ was dependent on the sex of the resistant progenitor. Sex ratio of the survivors to high doses of CryIA(b) in the F₁ of the two reciprocal crosses did not corroborate the preliminary hypothesis of resistance being due to a recessive sex-linked allele. Since, in a previous work, the loss of CryIA(b) binding capacity of resistant insects had been demonstrated, binding to midgut tissue sections from F₁ individuals was also analysed. The presence of binding in all of the F₁ preparations showed that, at least, a recessive autosomal allele was responsible for the loss of binding capacity in the resistant population.