Phenotypic,molecular and pathogenic characterization of Phlyctema vagabunda,causal agent of olive leprosy

Olive leprosy, caused by the fungus Phlyctema vagabunda, is a classic fruit rot disease widespread in the Mediterranean basin. From 2009 to 2013, new disease symptoms consisting of small circular necrotic leaf lesions, coin branch canker and shoot dieback were observed in Spanish and Portuguese olive orchards showing intense defoliation. Phlyctema‐like anamorphs were consistently isolated from leaves and shoots with symptoms. Representative isolates from affected leaves, shoots and fruits were characterized based on morphology of colonies and conidia, optimum growth temperature and comparison of DNA sequence data from four regions: ITS, tub2, MIT and rpb2. In addition, pathogenicity tests were performed on apple and olive fruits, and on branches and leaves of olive trees. Maximum mycelial growth rate ranged between 0.54 and 0.73 mm per day. Conidia produced on inoculated apple fruits showed slight differences in morphology among the representative fungal isolates evaluated. Phylogenetic analysis clustered all of the Phlyctema‐like isolates in the same clade, identifying them as Phlyctema vagabunda. On fruits, influence of wounding, ripening and cultivar resistance was studied, with cv. Blanqueta being the most susceptible cultivar. On branches, a mycelial‐plug inoculation method reproduced olive leprosy symptoms and caused shoot dieback. On leaves, Koch's postulates were fulfilled and the pathogen caused characteristic necrotic spots and plant defoliation. This is the first time that the pathogenicity of P. vagabunda in olive leaves has been demonstrated.     

<Emphasis Type="Italic">In vitro</Emphasis> larvicidal effect of a hydroalcoholic extract from <Emphasis Type="Italic">Acacia cochliacantha</Emphasis> leaf against ruminant parasitic nematodes

The aim of this study was to evaluate the in vitro lethal effect of a hydroalcoholic extract (HAE) from Acacia cochliacantha leaf against three gastrointestinal nematodes species (Haemonchus contortus, H. placei and Cooperia punctata) of domestic ruminants. The HAE was assessed using five concentrations: 100, 125, 175, 150 and 200 mg/ml; 0.5% Ivermectin was used as a positive control and distilled water, as negative control. The data were normalized using the square root and analysed with a completely randomized design through ANOVA analysis using the general lineal model (GLM) of the SAS program. The HAE tannin content was determined through spectrophotometry (UV-visible) and the other major phenols, were identified by chromatographic processes. The results showed an in vitro larvicidal activity of the HAE against the three assessed nematode species with all assessed concentrations. A clear HAE increased concentration dependence effect was observed. The highest activity of the HAE was obtained at the highest concentration (close to 100%, P < 0.05). This result was similar to the one obtained with Ivermectin. On the other hand, the chemical analysis of HAE showed the presence of tannins, caffeoyls and coumaroyl derivates and quercetin as the main compounds. The results suggest that the HAE from this plant species possess in vitro anthelmintic properties. The identified compounds in this study would good candidates for further in vivo researches.     

Effect of dominance-subordinate relationship and familiarity of an audience male on young rams' libido and semen characteristics

The purpose of this study was to investigate the range of circumstances under which the “audience effect” is expressed in rams, in particular, whether the dominance-subordinate relationship and familiarity of an audience male, influences young rams' libido and semen characteristics. To achieve this objective, semen from ten 10-month Katahdin rams was collected weekly in a vaginal collection vial, while the rams were exposed individually in each session to a dominant (familiar and unfamiliar) or subordinate (familiar and unfamiliar) male, or to no audience. Reaction time, semen volume, and sperm concentration were found to be similar (P > 0.05) in rams that were alone or in those that were being observed by an audience, regardless of the dominance-subordinate relationship or familiarity of the observer. The results of this experiment support the view that the presence and dominance-subordinate relationship of an audience regardless of their familiarity with the subject is irrelevant regarding the reaction time, semen volume, and sperm concentration of ram lambs during semen collection.     

Influence of tongue inspection during cysticercosis diagnosis on some behavioral and physiological stress measures in pigs (<Emphasis Type="Italic">Sus scrofa domestica</Emphasis>)

To evaluate the level of stress induced by the tongue inspection method during cysticercosis diagnosis in pigs at 60 and 90 kg, 27 animals were assigned to one of three experimental groups. In the first group (T1), pigs were held with a snout noose. In addition, animals in T2 were subjected to tongue inspection, while in T0, none of the procedures described above were performed. Resistance to be handled and serum cortisol concentration were higher (P < 0.05) than those from T0 in T1 and T2 groups, for 60- and 90-kg pigs. Time to return to the feeder observed the same pattern than previous variables in 60-kg pigs, but T1 and T0 were similar (P > 0.05) for the heavier animals. T2 did not build up a significant amount of stress with respect to T1, where only more (P > 0.05) vocalizations were emitted by 90-kg pigs. It was concluded that the use of snout noose induces major changes in some behavioral and physiological measures of stress in swine, while the opening of the mouth including tongue inspection does not contribute significantly to this effect.     

High prevalence of multiple resistance to antibiotics in Salmonella serovars isolated from a poultry slaughterhouse in Spain

Salmonellosis is a major foodborne infection in Spain, and strains that are resistant to a great variety of antibiotics have become a major public health concern. The aim of this study was to determine the level of antibiotic resistance in 133 Salmonella isolates obtained from a poultry slaughterhouse in Zaragoza (NE Spain). Antimicrobial resistance testing was performed by disk diffusion method using 19 antibiotics. Results were interpreted following the NCCLS criteria. Overall, the highest percentage of resistance was found to the following antimicrobial agents: sulfadiazine (96.2%), neomycin (53.4%), tetracycline (21.8%), and streptomycin (11.3%). All isolates were found to be resistant to one or more of the antibiotics tested. Multiple resistance was observed in 87 strains (65.4%). We found 23 different patterns of resistance in Salmonella Enteritidis. Resistance to sulfadiazine was the most common single resistance. The most frequent patterns of multiresistant strains were neomycin+sulfadiazine and neomycin+tetracycline+sulfadiazine. S. 4,5,12:b: showed the highest percentages of resistance to the tested drugs, with five different resistance patterns found. Ampicillin+chloramphenicol+streptomycin+sulphonamides+tetracycline (ACSSuT) resistance pattern, commonly associated with S. Typhimurium DT 104, was not detected in strains of the same phage type from broilers. The appearance of substantial multiresistance in foodborne Salmonella isolates suggests the need for more prudent use of antibiotics by farmers, veterinarians, and physicians.     

Analysis of fine-scale spatial pattern of a grassland from remotely-sensed imagery and field collected data

An important practical problem in the analysis of spatial pattern in ecological systems is that requires spatially-intensive data, with both fine resolution and large extent. Such information is often difficult to obtain from field-measured variables. Digital imagery can offer a valuable, alternative source of information in the analysis of ecological pattern. In the present paper, we use remotely-sensed imagery to provide a link between field-based information and spatially-explicit modeling of ecological processes. We analyzed one digitized color infrared aerial photograph of a serpentine grassland to develop a detailed digital map of land cover categories (31.24 m × 50.04 m of extent and 135 mm of resolution), and an image of vegetation index (proportional to the amount of green biomass cover in the field). We conducted a variogram analysis of the spatial pattern of both field-measured (microtopography, soil depth) and image-derived (land cover map, vegetation index, gopher disturbance) landscape variables, and used a statistical simulation method to produce random realizations of the image of vegetation index based upon our characterization of its spatial structure. The analysis revealed strong relationships in the spatial distribution of the ecological variables (e.g., gopher mounds and perennial grasses are found primarily on deeper soils) and a non-fractal nested spatial pattern in the distribution of green biomass as measured by the vegetation index. The spatial pattern of the vegetation index was composed of three basic components: an exponential trend from 0 m to 4 m, which is related to local ecological processes, a linear trend at broader scales, which is related to a general change in topography across the study site, and a superimposed periodic structure, which is related to the regular spacing of deeper soils within the study site. Simulations of the image of vegetation index confirmed our interpretation of the variograms. The simulations also illustrated the limits of statistical analysis and interpolations based solely on the semivariogram, because they cannot adequately characterize spatial discontinuities.     

A novel use of Nesidiocoris tenuis (Reuter) (Hemiptera: Miridae) as inoculative agent of baculoviruses

Background

Alphabaculoviruses are Lepidoptera-specific virulent pathogens that infect numerous pests, including the Spodoptera complex. Due to their low environmental persistence, the traditional use of Alphabaculoviruses as bioinsecticides consist in high-rate spray applications with repeated treatments. Several abiotic and biotic factors can foster its dispersion, promoting their persistence in the agroecosystem. Amongst biotic factors, predatory arthropods can disperse the viruses by excretion after preying on infected individuals. Therefore, this study focused on promoting predator's ingestion of nucleopolyhedrovirus (NPV)-treated diets, and the later exposition of the insect host to leaf surfaces contaminated with predator excreta. The virus–host–predator system studied was Spodoptera littoralis nucleopolyhedrovirus (SpliNPV), Spodoptera littoralis (Boisduval) and Nesidiocoris tenuis (Reuter). The infective potential of N. tenuis feces and the retention time of SpliNPV were assessed under laboratory conditions after feeding on treated diets (sucrose solution and Ephestia kuehniella eggs).

Results

Mortality of S. littoralis larvae was lower via N. tenuis excretion than in positive control (spray application) in the first infection cycle, together with a delay in host death. In the second infection cycle, both SpliNPV-treated diets triggered 100% mortality. Both diets allowed the transmission of SpliNPV, with a faster excretion via sucrose solution compared to E. kuehniella eggs. SpliNPV remained in N. tenuis digestive tract and was viable after excretion at least for 9 days for both diets.

Conclusions

This study demonstrated the potential of the predator N. tenuis as inoculative agent of baculoviruses, representing a new alternative that, along with inundative applications, might contribute to improve pest management strategies. © 2023 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.     

Upper alimentary tract papillomas in calves related to papillomavirus infection

This study reports 3 cases of spontaneous papillomavirus infection in 1-week-old calves. Thickening of the omasum and abomasum wall, with acute inflammation, necrosis, ulceration, and neoplastic changes were seen in 1 calf. In the other 2, small papillomas were observed in the omasal mucosa, exhibiting proliferation of the parakeratinized epithelium. Papillomavirus antigens were detected by immunohistochemistry and virus-like particles were seen through electron microscopy.     

Detection and quantification of Ilyonectria spp. associated with black‐foot disease of grapevine in nursery soils using multiplex nested PCR and quantitative PCR

Three nursery fields and three rootstock mother fields from commercial nurseries located in Comunidad Valenciana region (central‐eastern Spain) were surveyed in July 2011 to detect the presence and to quantify Ilyonectria spp. in the soil. In each field, ten soil samples were taken randomly with a soil probe at a depth of 10–30 cm, and 10–20 cm from the base of the plant. Three replicate subsamples (10 g each) were taken from each soil sample. DNA was extracted and a multiplex nested PCR with species‐specific primer pairs (Mac1/MaPa2, Lir1/Lir2 and Pau1/MaPa2) was used to identify the species present. Among the 180 soil DNA samples analysed, Ilyonectria spp. were detected in 172 of them. Ilyonectria macrodidyma complex was the most frequently detected, being identified in 141 samples from all the fields evaluated. However, I. liriodendri was detected in only 16 samples, but was present in all open‐root field nurseries and in two rootstock mother fields. In addition, quantitative PCR (qPCR) assays were done to assess the levels of I. liriodendri and I. macrodidyma‐complex DNA in the soil samples. Detection of Ilyonectria spp. DNA using qPCR correlated with the fields found positive with the nested multiplex PCR. DNA concentrations of Ilyonectria spp. ranged from 0·004 to 1904·8 pg μL^?1. In general, samples from rootstock mother fields showed the highest DNA concentrations. The ability to detect and quantify Ilyonectria spp. genomic DNA in soil samples from nursery fields and rootstock mother fields confirms soils from both field types as important inoculum sources for black‐foot pathogens.