Eugenol and menthol synergize the toxicity of permethrin in the blood-sucking bug, Triatoma infestans.

The blood-sucking bug, Triatoma infestans, is one of the main vectors of Chagas disease in America. It is usually controlled with pyrethroids, but the emergence of resistance to these insecticides creates the need to look for alternative products. Eugenol, menthol and menthyl acetate are botanical monoterpenes, which produce lethal and sublethal effects on insects. The purpose of this work was to determine what type of toxicological interactions occur when binary mixtures, formed by the pyrethroid permethrin and sublehtal doses of eugenol, menthol or menthyl acetate, are applied to T. infestans. First instar nymphs were exposed to filter papers impregnated with the insecticides. The number of knocked down insects was registered at different times and Knock Down Time 50% (KT50) values were calculated. The following KT50 values with their corresponding 95% Confidence Intervals were obtained: permethrin, 47.29 (39.92 - 56.32) min; permethrin + eugenol, 34.08 (29.60 - 39.01) min; permethrin + menthol, 27.54 (23.28 - 32.55) min; permethrin + menthyl acetate, 43.62 (39.99 - 47.59) min. Eugenol and menthol increased the speed of action of permethrin (synergism), but menthyl acetate had no effect on it (additivity). These results provide the basis to further explore interactions between conventional insecticides and plant monoterpenes as potential tools for controlling T. infestans.

The Behavioral Response of Blattella germanica (Blattodea: Ectobiidae) Exposed to DEET Varies Throughout Its Life Cycle.

The German cockroach, Blattella germanica L., is a hemimetabolous insect pest of economical and medical importance. N,N-diethyl-3-methylbenzamide (DEET) is an insect repellent whose effect on this species has received very little attention. The objective of this work was to determine whether the behavioral response of B. germanica to DEET varies along its life cycle. DEET repellence was assessed in small, medium, and large nymphs, and in adults of both sexes, all originated from the same laboratory colony (CIPEIN). The experimental arena consisted in a piece of filter paper treated with repellent on one half (195 µg/cm2) and solvent alone on the other half. A cockroach was placed on the filter paper, and its behavior was filmed. An image analyzer was used to quantify how long the insect spent on each side of the paper. As a control, a cockroach was exposed to a piece of filter paper treated with solvent (acetone) alone. Each assay was repeated independently six times. Distribution coefficient (DC) values were calculated, a parameter that ranges between 0 (attraction) and 1 (repellence). Small nymphs were more sensitive to DEET (mean DC = 0.93). The mean DC values of the other groups varied between 0.62 (medium nymphs) and 0.71 (male adults). The group of medium nymphs was the only one whose behavior was not significantly altered by exposure to DEET. The results show the importance of assessing insect repellents at different stages of the insect's life cycle in order to obtain a complete panorama of its effect.

Evaluating the impact of the biocontrol agent Trichoderma harzianum ITEM 3636 on indigenous microbial communities from field soils.

AIM: To investigate the impact of inoculating peanut seeds with the biocontrol agent Trichoderma harzianum ITEM 3636 on the structure of bacterial and fungal communities from agricultural soils. METHODS AND RESULTS: Polymerase chain reaction (PCR)-denaturing gradient gel electrophoresis (PCR-DGGE) and next-generation sequencing (NGS) of amplicons (or marker gene amplification metagenomics) were performed to investigate potential changes in the structure of microbial communities from fields located in a peanut-producing area in the province of Córdoba, Argentina. Fields had history of peanut smut (caused by Thecaphora frezii) incidence. The Shannon indexes (H'), which estimate diversity, obtained from the PCR-DGGE assays did not show significant differences neither for bacterial nor for fungal communities between control and inoculation treatments. On the other hand, the number of operational taxonomic units obtained after NGS was similar between all the analysed samples. Moreover, results of alpha and beta diversity showed that there were no significant variations between the relative abundances of the most representative bacterial and fungal phyla and genera, in both fields. CONCLUSIONS: Trichoderma harzianum ITEM 3636 decreases the incidence and severity of agriculturally relevant diseases without causing significant changes in the microbial communities of agricultural soils. SIGNIFICANCE AND IMPACT OF THE STUDY: Our investigations provide information on the structure of bacterial and fungal communities in peanut-producing fields after inoculation of seeds with a biocontrol agent.

Population genetic analysis and trichothecene profiling of Fusarium graminearum from wheat in Uruguay.

Fusarium graminearum sensu stricto (F. graminearum s.s.) is the major causal agent of Fusarium head blight of wheat worldwide, and contaminates grains with trichothecene mycotoxins that cause serious threats to food safety and animal health. An important aspect of managing this pathogen and reducing mycotoxin contamination of wheat is knowledge regarding its population genetics. Therefore, isolates of F. graminearum s.s. from the major wheat-growing region of Uruguay were analyzed by amplified fragment length polymorphism assays, PCR genotyping, and chemical analysis of trichothecene production. Of the 102 isolates identified as having the 15-ADON genotype via PCR genotyping, all were DON producers, but only 41 strains were also 15-ADON producers, as determined by chemical analysis. The populations were genotypically diverse but genetically similar, with significant genetic exchange occurring between them. Analysis of molecular variance indicated that most of the genetic variability resulted from differences between isolates within populations. Multilocus linkage disequilibrium analysis suggested that the isolates had a panmictic population genetic structure and that there is significant recombination occurs in F. graminearum s.s. In conclusion, tour findings provide the first detailed description of the genetic structure and trichothecene production of populations of F. graminearum s.s. from Uruguay, and expands our understanding of the agroecology of F. graminearum and of the correlation between genotypes and trichothecene chemotypes.

Diversity of black Aspergilli isolated from raisins in Argentina: Polyphasic approach to species identification and development of SCAR markers for Aspergillus ibericus.

Aspergillus section Nigri is a heterogeneous fungal group including some ochratoxin A producer species that usually contaminate raisins. The section contains the Series Carbonaria which includes the toxigenic species Aspergillus carbonarius and nontoxigenic Aspergillus ibericus that are phenotypically undistinguishable. The aim of this study was to examine the diversity of black aspergilli isolated from raisins and to develop a specific genetic marker to distinguish A. ibericus from A. carbonarius. The species most frequently found in raisins in this study were Aspergillus tubingensis (35.4%) and A. carbonarius (32.3%), followed by Aspergillus luchuensis (10.7%), Aspergillus japonicus (7.7%), Aspergillus niger (6.2%), Aspergillus welwitschiae (4.6%) and A. ibericus (3.1%). Based on inter-simple sequence repeat (ISSR) fingerprinting profiles of major Aspergillus section Nigri members, a sequence-characterized amplified region (SCAR) marker was identified. Primers were designed based on the conserved regions of the SCAR marker and were utilized in a PCR for simultaneous identification of A. carbonarius and A. ibericus. The detection level of the SCAR-PCR was found to be 0.01 ng of purified DNA. The present SCAR-PCR is rapid and less cumbersome than conventional identification techniques and could be a supplementary strategy and a reliable tool for high-throughput sample analysis.

Development of amplified fragment length polymorphism (AFLP)-derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot.

AIMS: The objective of this work was to design an amplified fragment length polymorphism (AFLP)-derived specific primer for the detection of Fusarium solani aetiological agent of peanut brown root rot (PBRR) in plant material and soil. METHODS AND RESULTS: Specific primers for the detection of the pathogen were designed based on an amplified region using AFLPs. The banding patterns by AFLPs showed that isolates from diseased roots were clearly distinguishable from others members of the F. solani species complex. Many bands were specific to F. solani PBRR, one of these fragments was selected and sequenced. Sequence obtained was used to develop specific PCR primers for the identification of pathogen in pure culture and in plant material and soil. Primer pair FS1/FS2 amplified a single DNA product of 175 bp. Other fungal isolates occurring in soil, included F. solani non-PBRR, were not detected by these specific primers. The assay was effective for the detection of pathogen from diseased root and infected soils. CONCLUSIONS: The designed primers for F. solani causing PBRR can be used in a PCR diagnostic protocol to rapidly and reliably detect and identify this pathogen. SIGNIFICANCE AND IMPACT OF THE STUDY: These diagnostic PCR primers will aid the detection of F. solani causing PBRR in diseased root and natural infected soils. The method developed could be a helpful tool for epidemiological studies and to avoid the spread of this serious disease in new areas.

Natural deoxynivalenol occurrence and genotype and chemotype determination of a field population of the Fusarium graminearum complex associated with soybean in Argentina.

Soybean (Glycine max L.), the main source of protein throughout the world, is used both as a food and a feedstuff. Currently, limited information about the occurrence of Fusarium species and mycotoxins in soybean grain and by-products is available. The aims of the present study were: (1) to identify toxigenic Fusarium species associated with soybean during crop reproductive stages; (2) to determine the occurrence of deoxynivalenol (DON) and nivalenol (NIV) in soybean seeds; (3) to determine the genotype and chemotype of selected Fg complex strains using molecular and chemical analysis, respectively; and (4) to characterize the strains using AFLP(s) markers. One soybean field located at Córdoba Province, Argentina, was monitored and samples of soybean tissue were harvested at three reproductive stages: flowering (R2), full seed (R6) and full maturity (R8). A total of 389 Fusarium strains F. equiseti (40%) was the most frequently species recovered followed by F. semitectum (27%) and F. graminearum (Fg) (11%). From the 40 soybean samples analysed, only two presented detectable DON levels. Based on DON occurrence on soybean seeds at ripening stages, the toxigenic ability of Fg complex strains isolated from soybean seeds, pods and flowers were analysed. The trichothecene genotype was determined by a multiplex PCR using primers based on Tri3, Tri5 and Tri7 toxin genes and then the chemotype was verified by chemical analysis. Most Fg complex strains showed 15-ADON genotype and five strains presented a DON/NIV; these also produced both toxins under in vitro culture. Neither the NIV nor the 3-ADON genotypes were detected among the members of the population evaluated. All the 15-ADON genotype strains were characterized as F. graminearum sensu stricto (lineage 7), while the strains presented a DON/NIV genotype were characterized as F. meridionale (lineage 2). The present study contributes new information on the occurrence of Fusarium species and trichothecenes toxins on soybean at the pre-harvest stages. Also, this is the first report on the chemotype, genotype and lineages among Fg complex isolated from soybean.

Molecular characterization and toxigenic profile of Aspergillus section Nigri populations isolated from the main grape-growing regions in Argentina.

AIMS: The objective of this study was to evaluate the biodiversity of Aspergillus section Nigri populations from Argentinean vineyards by morphological, toxigenic and AFLP analysis. MATERIALS AND METHODS: Five hundred and thirty-eight strains were isolated from grapes during 2006/07 and 2007/08 vintages. The morphological identification and toxigenic profile for all strains isolated were performed. Eighty-eight strains were selected for characterization at species level by AFLP markers. Cluster analysis showed a clear separation into four main groups: A. carbonarius, A. tubingensis, A. niger'aggregate' and Aspergillus'uniseriate'. A. carbonarius strains constituted a homogeneous group, while a high degree of genetic diversity was found within the A. niger'aggregate' and 'A. uniseriate' clusters. The A. tubingensis cluster was the most prevalent group and was clearly separated from A. niger'aggregate'. Ten strains showed 45% homology with A. tubingensis FRR 5720 ex-type strain and were considered as 'atypical' or a closely related species. AFLP results indicate that no genotypical differences can be established between ochratoxigenic and nonochratoxigenic strains. CONCLUSIONS: Aspergillus section Nigri populations on grapes were represented mainly by four groups. A. tubingensis species were separated from A. niger'aggregate' group and some of their strains produced OTA. SIGNIFICANCE AND IMPACT OF THE STUDY: This study provides new data on molecular characterization of Aspergillus section Nigri populations in Argentina.

Population genetic structure of Gibberella zeae isolated from wheat in Argentina.

Gibberella zeae (anamorph Fusarium graminearum) causes Fusarium head blight of wheat. The authors used amplified fragment length polymorphisms (AFLPs) to characterize the genetic structure of two G. zeae populations from commercial wheat fields. The working hypothesis was that sufficient genetic exchange occurs between local populations to prevent significant partitioning of allelic variation. We analysed 216 AFLP loci for 113 isolates collected during the 2002 harvest season. All strains had AFLP profiles typical of G. zeae lineage 7. Both populations were genotypically diverse but genetically similar and potentially part of a larger, randomly mating population, with significant genetic exchange probably occurring between the two subpopulations. Linkage disequilibrium was low, but higher than reported for many other populations of G. zeae, and about 20% of the alleles detected were specific to one of the two subpopulations - results consistent with limited gene exchange between the two subpopulations. This study extends previous work with populations of G. zeae to include those found in Argentina, one of the world's largest wheat growing countries.

Molecular characterization of Aspergillus section Flavi isolates collected from peanut fields in Argentina using AFLPs.

AIMS: The objectives of this study were: (i) to evaluate genetic relatedness among Aspergillus section Flavi strains isolated from soil and peanut seeds in Argentina; (ii) to determine if AFLP molecular markers could be useful to identify isolates up to species level, and to correlate these markers with the isolates' toxigenic potentials and/or vegetative compatibility group (VCG) affiliations. METHODS AND RESULTS: Amplified fragment length polymorphism (AFLPs) analysis was applied to compare 82 isolates of Aspergillus section Flavi. Cluster analysis showed a clear separation of A. flavus and A. parasiticus, and comparison of fingerprints revealed several specific markers for each group of isolates. AFLP analysis indicates that no genotypical differences can be established between aflatoxigenic and nonaflatoxigenic producers in both species analysed. In addition, candidate AFLP markers associated with a particular VCG were not found. CONCLUSIONS: There was a concordance between morphological identification and separation up to species level using molecular markers. The findings of specific bands for A. flavus and A. parasiticus may be useful for the design of specific PCR primers in order to differentiate these species and detect them in food. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study provides new data on molecular characterization of Aspergillus section Flavi in Argentina.

Fusarium species (section Liseola) and its mycotoxins in maize harvested in northern Argentina.

Maize and maize products harvested in small fields and stored by farmers in northern Argentina were assayed for Fusarium and fumonisin and beauvericin contamination. Fumonisins were present in six of the 18 samples. The levels of fumonisins ranged from 603 to 1888 ng/kg. Fumonisin B3 (FB3) and beauvericin were not detected in the samples evaluated. Fusarium subglutinans was one of the most prevalent species isolated. Twenty-five strains of F. subglutinans isolated from maize kernels and belonging to Gibberella fujikuroi mating population E were beauvericin-producers in culture. Seven of these strains also produced moniliformin. This is the first report on beauvericin-production by maize isolates of F. subglutinans from Argentina.

Mycoflora and naturally occurring mycotoxins in poultry feeds in Argentina.

The purpose of this work was to determine the mycoflora and mycotoxins natural incidence in poultry feeds from 2 factories in Río Cuarto, Córdoba. One hundred and thirty samples were taken from May/1996 to May/1997. The most dominant species isolated of poultry feed samples belonged to the genera Aspergillus spp 85% and Fusarium spp 70%. From Aspergillus genus eleven species were identified and A. flavus was the most frequent. Nine species were identified from the Fusarium genus and the predominant was F. moniliforme. Penicillium ranked third in the number of isolated cases. From this genus twelve species were collected of which P. brevicompactum (15%), P. restrictum (14%) and P. purpurogenum (12%) were the most common. The most significant mycotoxin from poultry feeds was aflatoxin B1 (AFB1) found in 48% of the samples, with levels ranging from 10 to 123 ng/g. For zearalenone (ZEA) the levels were 327 to 5, 850 ng/g and DON was not detected from the samples. Due to the fact that in Argentina there is little information about this topic, these data on poultry feeds in our region would be of worldwide interest.

Natural occurrence of fumonisins and their correlation to Fusarium contamination in commercial corn hybrids growth in Argentina.

Fifty commercial corn hybrids with different endosperm characteristics, vegetative cycle length and cross class grown in the same geographical area (Cordoba Province, Argentina) were analysed for fumonisin accumulation. All hybrids analysed showed fumonisin B(1) and B(2) contamination ranging from 185 to 27,050 ng/g for FB(1) and from 40 to 9950 ng/g for FB(2). Although most of the hybrids analysed had flint-type endosperm, two hybrids with dent-type endosperm (e.g. Prozea 10 and AX 746) showed the highest level of fumonisin (37,000 ng/g) and more FB(2) than FB(1) (FB(2)/FB(1) ratio 2.42), respectively. There was no correlation between fumonisin concentration and length of the vegetative cycle. Among 18 hybrids examined for Fusarium species contamination there was also no correlation between fumonisin contamination and the level of infection with Fusarium species (Section Liseola). Eighteen hybrids showed fumonisin levels lower than 1000 ng/g. This result suggests that there is some possibility of selecting hybrids resistant or less susceptible to fumonisin and Fusarium contamination.