In vitro compatibility of Brassicaceae extracts with nematophagous fungi and their effects against Nacobbus celatus.

Nacobbus celatus sp. n. is one of the main root-knot nematodes in the field destined for horticultural production of the central region of Argentine due to its ability to infect several host plants. The lack of new and safe active ingredients against this nematode has restricted control alternatives for growers. Egg-parasitic fungi and biofumigation with brassicaceae have been considered as potential candidates for the development of bionematicides. Nematicidal effects of Brassica oleracea var. italica (broccoli) and Brassica oleracea var. capitata (cabbage) aqueous extracts (AEs) against second-stage juveniles (J2) of N. celatus were evaluated in vitro. Fisher LSD tests evidenced significant nematicidal (α = 0.05) effects of the two AEs tested, with LD100 of 250 and 500 µL mL-1 for broccoli and cabbage, respectively. Compatibility assays between AEs and five nematophagous fungi were performed on soil extract medium conditioned at 0.99 water activity and incubated at 30, 25 and 20 °C. Purpureocillium lilacinum SR14 was the fungal strain that showed compatibility at levels of spore viability, growth rate and conidia productions at LD50 (125 µL mL-1) and LD25 (60 µL mL-1) of broccoli aqueous extract (BAE) and enhanced the nematophagous effect. Moreover, phytotoxic studies revealed that 125 µL mL-1 of BAE applied at the transplantation time could be safely used without affecting tomato culture. In conclusion, the integrated application of BAE with P. lilacinum SR14, which combines two action mechanisms, represents a promising integrated strategy to management phytoparasitic nematodes.

In vitro characterization bioassays of the nematophagous fungus Purpureocillium lilacinum: Evaluation on growth, extracellular enzymes, mycotoxins and survival in the surrounding agroecosystem of tomato.

The effects of water stress and temperature on in vitro growth and enzymatic activity of Purpureocillium lilacinum (Sordariomycetes, Hypocreales, Ophiocordycipitaceae) isolates with demonstrated capacity to control Nacobbus aberrans (Secernentea, Tylenchida, Pratylenchidae) were evaluated in this study. Also, saprophytic and endophytic colonization in tomato plants were determined. P. lilacinum was able to grow under the evaluated levels of osmotic and matric stress, but the increase in water stress caused reductions in radial growth rates. Moreover, the fungal isolates produced chitinases, proteases, and leucinostatins under inductive conditions. The nematophagous fungi were able to develop saprophytically (104 CFU g-1 of soil). Meanwhile, only P. lilacinum SR38 demonstrated endophytic capacity. The results suggest that P. lilacinum can be effectively applied as biocontrol agents of phytoparasitic nematodes in tomatoes under variable agroecological conditions.

Microencapsulated food-grade antioxidant applied as a preservative of peanut seed quality in microcosm- and pilot-scale trials.

BACKGROUND: In Argentina, peanuts are stored for 3-6 months. It is important to avoid proliferation of fungi and insect pests during this period. In this study, the potential of butylated hydroxyanisole (BHA) microcapsules to conserve peanut kernels was evaluated in microcosms and on a pilot scale. RESULTS: In microcosm assays, microcapsules containing BHA at a dose of 1802 µg g-1 reduced 37% of total fungal count. Higher reductions (77-100%) were obtained with a combined treatment with BHA formulation (1802 µg g-1 ) plus fungicide (methyl thiophanate 0.0100 g L-1  and metalaxyl 0.0133 g L-1 ). However, germination levels of peanut seeds treated with the BHA formulation were less than 6% throughout the incubation time. In pilot-scale trials, the storage conditions allowed the control of fungal development and insect proliferation. Quantifiable levels of BHA were also detected throughout the entire storage period. The combined treatment significantly reduced fungal contamination at 2 months of storage (C1-2015: 37.41%; C1-2016: 28.48%; C2-2016: 45.02%). Seed germination of unshelled stored peanuts was not affected by the formulation. CONCLUSION: The application of the BHA formulation during storage combined with pre-seeding treatment could be an appropriate strategy to maintain the quality of the peanut kernels destined for seed. © 2018 Society of Chemical Industry.

Effect of micro-encapsulated antioxidant formulations on mycobiota, residual levels, sensory analyses and insect pest attack in stored peanuts.

The in situ effect of microencapsulated 2(3)-tert-butyl-4 hydroxyanisole (BHA) on stored peanuts (Arachis hipogaea) intended for human consumption was evaluated. Peanut were stored unshelled in flexible containers called "big bags" that were made of polypropylene raffia. 100 kg of peanuts were used in each big bag and stored in refrigerated cells (<18 °C) for about 5 months in two different peanut processing companies during 2015/2016 period. Fungal populations, aflatoxin accumulation, BHA residues, acidity and fatty acid profile, sensory analyses, insect damage and environmental factors variation, were evaluated. At the end of the storage period, significant (p < 0.05) fungitoxic effects of the BHA formulation were observed in the order of 30 and 15% for the first and second company, respectively. Cladosporium, yeasts, Penicillium, Fusarium, Alternaria and Aspergillus were the main fungal isolates. No aflatoxins were found for both companies and years evaluated. In addition, taste of the peanuts was not significantly affected (p < 0.05) by formulation used and insect damage was always lower than 3%. However, different levels of BHA were detected throughout the experiment in the two companies, with final levels of 2.5 for the C1 and 275 ng BHA/g peanuts in C2. Formulation did not affect acidity and organoleptic properties of peanuts. These results show that BHA formulation could be used as part of alternative strategy for control of fungal contamination storage period.

Screening and identification of horticultural soil fungi for their evaluation against the plant parasitic nematode Nacobbus aberrans.

The plant-parasitic nematode Nacobbus aberrans is an endoparasite causing severe losses to a wide range of crops from North to South America. The use of native antagonistic fungi may be considered as a possible biological control alternative to reduce the damages caused by this species. Antagonistic effects of 66 potential nematophagous fungi against eggs (J1) and second-stage juveniles (J2) of N. aberrans, were evaluated in vitro on water agar. DGC test showed significant differences (p < 0.0001) in the efficacy of some fungal isolates tested, with parasitism levels for J1 and J2 of 0-95 and 1-78%, respectively. Five isolates of Purpureocillium lilacinum, Metarhizium robertsii and Plectosphaerella plurivora appeared as the most effective antagonists of N. aberrans, relying on hyphae and adhesive conidia in host infection processes.

Efficacy of epiphytic bacteria to prevent northern leaf blight caused by Exserohilum turcicum in maize / Eficacia de bacterias epifíticas en la prevención del tizón foliar del maíz causado por Exserohilum turcicum

Eight potential biological control agents (BCAs) were evaluated in planta in order to assess their effectiveness in reducing disease severity of northern leaf blight caused by Exserohilum turcicum. The assay was carried out in greenhouse. Twenty-six-day-old plants, V4 phenological stage, were inoculated with antagonists by foliar spray. Only one biocontrol agent was used per treatment. Ten days after this procedure, all treatments were inoculated with E. turcicum by foliar application. Treatments performed were: C-Et: control of E. turcicum; T1: isolate 1 (Enterococcus genus) + E. turcicum; T2: isolate 2 (Corynebacterium genus) + E. turcicum; T3: isolate 3 (Pantoea genus) + E. turcicum; T4: isolate 4 (Corynebacterium genus) + E. turcicum; T5: isolate 5 (Pantoea genus) + E. turcicum; T6: isolate 6 (Bacillus genus) + E. turcicum; T7: isolate 7 (Bacillus genus) + E. turcicum; T8: isolate 8 (Bacillus genus) + E. turcicum. Monitoring of antagonists on the phyllosphere was performed at different times. Furthermore, the percentage of infected leaves and, plant and leaf incidence were determined. Foliar application of different bacteria significantly reduced the leaf blight between 30-78% and 39-56% at 20 and 39 days respectively. It was observed that in the V10 stage of maize plants, isolate 8 (Bacillus spp.) caused the greatest effect on reducing the severity of northern leaf blight. Moreover, isolate 8 was the potential BCA that showed more stability in the phyllosphere. At 39 days, all potential biocontrol agents had a significant effect on controlling the disease caused by E. turcicum.

Se evaluó a 8 potenciales agentes de control biológico (ACB) en un ensayo in planta, con el objetivo de probar su efectividad en la reducción del daño provocado por Exserohilum turcicum, agente causal del tizón foliar del maíz. El ensayo se llevó a cabo en invernadero. Plantas de maíz de 26 días, en estadio fenológico V4, se inocularon con los potenciales antagonistas por aplicación foliar como espray. Solo un agente de biocontrol fue usado por tratamiento y todos los tratamientos se inocularon con E. turcicum 10 días después, también por aplicación foliar. Los tratamientos desarrollados fueron los siguientes: C-Et: control de E. turcicum; T1: aislamiento 1 (género Enterococcus) + E. turcicum; T2: aislamiento 2 (género Corynebacterium) + E. turcicum; T3: aislamiento 3 (género Pantoea) + E. turcicum; T4: aislamiento 4 (género Corynebacterium) + E. turcicum; T5: aislamiento 5 (género Pantoea) + E. turcicum; T6: aislamiento 6 (género Bacillus) + E. turcicum; T7: aislamiento 7 (género Bacillus) + E. turcicum; T8: aislamiento 8 (género Bacillus) + E. turcicum. La monitorización en la filosfera de los antagonistas se llevó a cabo a diferentes tiempos. Además, se determinó el porcentaje de hojas infectadas y la incidencia en plantas y hojas. La aplicación foliar de diferentes bacterias redujo significativamente la gravedad del tizón del maíz: entre el 30 y el 78% a los 20 días y entre el 39 y el 56% a los 39 días. En el estadio V10 de las plantas de maíz se observó que el aislamiento 8 (Bacillus spp.) causó el mayor efecto de reducción del tizón foliar. Además, dicho aislamiento fue el potencial agente de biocontrol que mostró mayor estabilidad en la filosfera. A los 39 días, todos los potenciales agentes de biocontrol demostraban un efecto significativo sobre el control de la enfermedad causada por E. turcicum.

Efficacy of epiphytic bacteria to prevent northern leaf blight caused by Exserohilum turcicum in maize.

Eight potential biological control agents (BCAs) were evaluated in planta in order to assess their effectiveness in reducing disease severity of northern leaf blight caused by Exserohilum turcicum. The assay was carried out in greenhouse. Twenty-six-day-old plants, V4 phenological stage, were inoculated with antagonists by foliar spray. Only one biocontrol agent was used per treatment. Ten days after this procedure, all treatments were inoculated with E. turcicum by foliar application. Treatments performed were: C-Et: control of E. turcicum; T1: isolate 1 (Enterococcus genus)+E. turcicum; T2: isolate 2 (Corynebacterium genus)+E. turcicum; T3: isolate 3 (Pantoea genus)+E. turcicum; T4: isolate 4 (Corynebacterium genus)+E. turcicum; T5: isolate 5 (Pantoea genus)+E. turcicum; T6: isolate 6 (Bacillus genus)+E. turcicum; T7: isolate 7 (Bacillus genus)+E. turcicum; T8: isolate 8 (Bacillus genus)+E. turcicum. Monitoring of antagonists on the phyllosphere was performed at different times. Furthermore, the percentage of infected leaves and, plant and leaf incidence were determined. Foliar application of different bacteria significantly reduced the leaf blight between 30-78% and 39-56% at 20 and 39 days respectively. It was observed that in the V10 stage of maize plants, isolate 8 (Bacillus spp.) caused the greatest effect on reducing the severity of northern leaf blight. Moreover, isolate 8 was the potential BCA that showed more stability in the phyllosphere. At 39 days, all potential biocontrol agents had a significant effect on controlling the disease caused by E. turcicum.

Improvement of the Insecticidal Capacity of Two Purpureocillium Lilacinum Strains against Tribolium Confusum.

Entomopathogenic fungi can regulate insect populations. They have extracellular enzymes that degrade cuticle components, mainly hydrocarbons, used as an energy source. The increase in insecticidal activity of fungi in a medium supplemented with cuticular hydrocarbons was assayed and the hydrolytic enzyme profiles of two strains of Purpureocillium lilacinum were evaluated. A spore suspension of P. lilacinum was inoculated in Petri plates with different values (0.99-0.97-0.95) of water activity (Aw) using the substrates gelatin, starch and tween-20. Growth rate on the different substrates and the enzymatic activity index for proteases, amylases and lipases at different incubation times, pH and Aw, was evaluated. Moreover, the insecticidal efficiency of strains grown in media supplemented with n-hexadecane and n-octacosane was analyzed. LT50 was calculated against adults of Tribolium confusum and showed that mortality increased about 15% when the strains grew in amended culture medium. High amylolytic activity was detected, but proteases were the main enzymes produced. Optimal protease production was observed in a range of acid and alkaline pH and lower Aw. The greatest growth rate was obtained in presence of gelatin. Lipase and amylase production was detected in small amounts. Fungal growth in media with hydrocarbon mixtures increased the pathogenicity of the two strains of P. lilacinum, with the strain JQ926223 being more virulent. The information obtained is important for achieving both an increase in insecticidal capacity and an understanding of physiological adaptation of the fungus.

Selection of potential biological control of Exserohilum turcicum with epiphytic microorganisms from maize.

The aims of this study were to select microbial isolates from phyllosphere of maize and to examine their antagonistic activity against Exserohilum turcicum. Selection was performed through the ability of isolates to compete with the pathogen using an index of dominance and to affect growth parameters of E. turcicum. Most of the epiphytic populations obtained for the screening were bacteria. These isolates were found in the order of 6 log CFU/g of leaf fresh weight. According to similar morphological characteristics and staining, 44 out of 111 isolates obtained were selected for testing antagonistic effects. At water potential, ψ, -1.38MPa and -4.19MPa, three Bacillus isolates showed dominance at a distance (5/0) and a significant reduction of growth rate of the pathogen. Three Bacillus isolates only decreased the growth rate of E. turcicum at -1.38MPa. At -4.19MPa the growth rate decreased with three isolates of Pantoea and three Bacillus. In this study a negative and significant correlation was observed between the growth rate of E. turcicum and the dominance index in the interaction of the pathogen with some bacteria. These results show that with decreasing growth rate of the pathogen the dominance index of the interaction increases. Eleven potential biocontrol agents against E. turcicum were selected.

Selection of potential biological control of Exserohilum turcicum with epiphytic microorganisms from maize / Selección de microorganismos epifíticos de maíz como potenciales agentes de biocontrol de Exserohilum turcicum

The aims of this study were to select microbial isolates from phyllosphere of maize and to examine their antagonistic activity against Exserohilum turcicum. Selection was performed through the ability of isolates to compete with the pathogen using an index of dominance and to affect growth parameters of E. turcicum. Most of the epiphytic populations obtained for the screening were bacteria. These isolates were found in the order of 6 log CFU/g of leaf fresh weight. According to similar morphological characteristics and staining, 44 out of 111 isolates obtained were selected for testing antagonistic effects. At water potential, ψ, −1.38 MPa and −4.19 MPa, three Bacillus isolates showed dominance at a distance (5/0) and a significant reduction of growth rate of the pathogen. Three Bacillus isolates only decreased the growth rate of E. turcicum at −1.38 MPa. At −4.19 MPa the growth rate decreased with three isolates of Pantoea and three Bacillus. In this study a negative and significant correlation was observed between the growth rate of E. turcicum and the dominance index in the interaction of the pathogen with some bacteria. These results show that with decreasing growth rate of the pathogen the dominance index of the interaction increases. Eleven potential biocontrol agents against E. turcicum were selected.

El objetivo de este estudio fue seleccionar aislamientos microbianos de la filósfera de maíz y examinar su actividad antagonista contra Exserohilum turcicum. La selección se realizó a través de la capacidad de los aislamientos de competir con el patógeno usando un índice de dominancia y también la capacidad de afectar los parámetros de crecimiento de E. turcicum. La mayoría de las poblaciones epifíticas aisladas para la selección fueron bacterias. Estos aislamientos se encontraron en el orden de 6 log de UFC por gramo de peso fresco de hoja de maíz. En base a características morfológicas y tintóreas similares, se seleccionaron 44 de 111 aislamientos obtenidos para evaluar su capacidad antagónica. A los potenciales agua, ψ, −1,38 MPa y −4,19 MPa, tres aislados del género Bacillus mostraron dominancia a distancia (5/0) y una reducción significativa de la velocidad de crecimiento del patógeno. Tres aislamientos de Bacillus disminuyeron la velocidad de crecimiento de E. turcicum a −1,38 MPa. A −4,19 MPa la velocidad de crecimiento disminuyó con tres aislamientos de Pantoea y tres de Bacillus. En este estudio se observó una correlación negativa y significante entre la velocidad de crecimiento de E. turcicum y el índice de dominancia cuando el patógeno interactuó con algunas bacterias. Esto estaría indicando que cuando disminuye la velocidad de crecimiento del patógeno se incrementa el índice de dominancia de la interacción. Se seleccionaron once posibles agentes de biocontrol contra E. turcicum.

Selection of potential biological control of Exserohilum turcicum with epiphytic microorganisms from maize / Selección de microorganismos epifíticos de maíz como potenciales agentes de biocontrol de Exserohilum turcicum

The aims of this study were to select microbial isolates from phyllosphere of maize and to examine their antagonistic activity against Exserohilum turcicum. Selection was performed through the ability of isolates to compete with the pathogen using an index of dominance and to affect growth parameters of E. turcicum. Most of the epiphytic populations obtained for the screening were bacteria. These isolates were found in the order of 6 log CFU/g of leaf fresh weight. According to similar morphological characteristics and staining, 44 out of 111 isolates obtained were selected for testing antagonistic effects. At water potential, ψ, −1.38 MPa and −4.19 MPa, three Bacillus isolates showed dominance at a distance (5/0) and a significant reduction of growth rate of the pathogen. Three Bacillus isolates only decreased the growth rate of E. turcicum at −1.38 MPa. At −4.19 MPa the growth rate decreased with three isolates of Pantoea and three Bacillus. In this study a negative and significant correlation was observed between the growth rate of E. turcicum and the dominance index in the interaction of the pathogen with some bacteria. These results show that with decreasing growth rate of the pathogen the dominance index of the interaction increases. Eleven potential biocontrol agents against E. turcicum were selected.(AU)

El objetivo de este estudio fue seleccionar aislamientos microbianos de la filósfera de maíz y examinar su actividad antagonista contra Exserohilum turcicum. La selección se realizó a través de la capacidad de los aislamientos de competir con el patógeno usando un índice de dominancia y también la capacidad de afectar los parámetros de crecimiento de E. turcicum. La mayoría de las poblaciones epifíticas aisladas para la selección fueron bacterias. Estos aislamientos se encontraron en el orden de 6 log de UFC por gramo de peso fresco de hoja de maíz. En base a características morfológicas y tintóreas similares, se seleccionaron 44 de 111 aislamientos obtenidos para evaluar su capacidad antagónica. A los potenciales agua, ψ, −1,38 MPa y −4,19 MPa, tres aislados del género Bacillus mostraron dominancia a distancia (5/0) y una reducción significativa de la velocidad de crecimiento del patógeno. Tres aislamientos de Bacillus disminuyeron la velocidad de crecimiento de E. turcicum a −1,38 MPa. A −4,19 MPa la velocidad de crecimiento disminuyó con tres aislamientos de Pantoea y tres de Bacillus. En este estudio se observó una correlación negativa y significante entre la velocidad de crecimiento de E. turcicum y el índice de dominancia cuando el patógeno interactuó con algunas bacterias. Esto estaría indicando que cuando disminuye la velocidad de crecimiento del patógeno se incrementa el índice de dominancia de la interacción. Se seleccionaron once posibles agentes de biocontrol contra E. turcicum.(AU)

Selection of potential biological control of Exserohilum turcicum with epiphytic microorganisms from maize. / Selection of potential biological control of Exserohilum turcicum with epiphytic microorganisms from maize.

The aims of this study were to select microbial isolates from phyllosphere of maize and to examine their antagonistic activity against Exserohilum turcicum. Selection was performed through the ability of isolates to compete with the pathogen using an index of dominance and to affect growth parameters of E. turcicum. Most of the epiphytic populations obtained for the screening were bacteria. These isolates were found in the order of 6log CFU/g of leaf fresh weight. According to similar morphological characteristics and staining, 44 out of 111 isolates obtained were selected for testing antagonistic effects. At water potential, ¤ê, -1.38MPa and -4.19MPa, three Bacillus isolates showed dominance at a distance (5/0) and a significant reduction of growth rate of the pathogen. Three Bacillus isolates only decreased the growth rate of E. turcicum at -1.38MPa. At -4.19MPa the growth rate decreased with three isolates of Pantoea and three Bacillus. In this study a negative and significant correlation was observed between the growth rate of E. turcicum and the dominance index in the interaction of the pathogen with some bacteria. These results show that with decreasing growth rate of the pathogen the dominance index of the interaction increases. Eleven potential biocontrol agents against E. turcicum were selected.

Antifungal impact of volatile fractions of Peumus boldus and Lippia turbinata on Aspergillus section Flavi and residual levels of these oils in irradiated peanut.

To investigate the antifungal properties of essential oil (EO) vapors from boldo and poleo on Aspergillus section Flavi and the residual levels of the oils in peanut, irradiated peanuts conditioned at three water activities (0.98, 0.95, 0.93) were treated with 2 and 3 µL/g of boldo and 3 and 5 µL/g of poleo. EO treatments produced the greatest impact on fungal growth parameters, followed by oil concentrations and aW levels. The three main components in peanut exposed to oil vapors were piperitone oxide, α-terpinene and eucalyptol for boldo and ß-caryophyllene epoxide, limonene and piperitenone for poleo. Residues of boldo and poleo EO were significantly decreased from 24.7 to 100% and from 26.6 to 99.7% at the end of the incubation period, respectively. The application of nontoxic boldo oil as fumigant in the control of Aspergillus section Flavi may represent a potential alternative antifungal treatment, without significant residues after 35 days.

Evaluation of the control ability of five essential oils against Aspergillus section Nigri growth and ochratoxin A accumulation in peanut meal extract agar conditioned at different water activities levels.

Essential oils (EOs) from boldo [Pëumus boldus Mol.], poleo [Lippia turbinata var. integrifolia (Griseb.)], clove [Syzygium aromaticum L.], anise [Pimpinella anisum] and thyme [Thymus vulgaris]) obtained by hydrodistillation were evaluated for their effectiveness against the growth of Aspergillus niger aggregate and A. carbonarius and accumulation of ochratoxin A (OTA). The evaluation was performed by compound dissolution at the doses of 0, 500, 1500 and 2500µL/L in peanut meal extract agar (PMEA) and exposure to volatiles of boldo, poleo (0, 1000, 2000 and 3000µL/L) and clove oils (0, 1000, 3000 and 5000µL/L), taking into account the levels of the water activity of the medium (a(W) 0.98, 0.95, 0.93). Statistical analyses on growth of Aspergillus strains indicated that the major effect was produced by oil concentrations followed by substrate a(W), and that reductions in antifungal efficiency of the oils tested were observed in vapor exposure assay. At all a(W) levels, complete fungal growth inhibition was achieved with boldo EO at doses of 1500 and 2000µL/L by contact and volatile assays, respectively. Contact exposure by poleo and clove EOs showed total fungal inhibition at the middle level tested of 1500µL/L, regardless of a(W), while their antifungal effects in headspace volatile assay were closely dependent on medium a(W). The fumigant activity of poleo (2000µL/L) and clove oils (3000µL/L) inhibited growth rate by 66.0% and 80.6% at a(W) 0.98 and 0.93, respectively. OTA accumulation was closely dependent on a(W) conditions. The antiochratoxigenic property of the volatile fractions of boldo, poleo and clove EOs (1000µL/L) was more significant at low a(W) levels, inhibition percentages were estimated at 14.7, 41.7 and 78.5% at a(W) 0.98, 0.95 and 0.93, respectively. Our results suggest that boldo, poleo and clove oils affect the OTA biosynthesis pathway of both Aspergillus species. This finding leaves open the possibility of their use by vapor exposure as effective non-toxic biopreservatives against OTA contamination in stored peanuts.

Influence of sub-lethal antioxidant doses, water potential and temperature on growth, sclerotia, aflatoxins and aflD (=nor-1) expression by Aspergillus flavus RCP08108.

Effects of interacting conditions of sub-lethal levels of antioxidants, water potential (Ψ) and temperature were evaluated on growth, sclerotial characteristics, aflatoxin B(1) (AFB(1)) production and aflD (=nor-1) gene expression by Aspergillus flavus strain RCP08108. These studies were carried out on peanut meal extract agar osmotically modified to -2.8,-7.1, -9.9 and -16.0 MPa and incubated at 28 and 20°C. The food grade antioxidants added were butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) at (1+1 mM-M1) and (5+5 mM-M2). To relate the aflD expression after toxigenic A. flavus grew under interacting stress conditions, real-time PCR was used. Antioxidant mixtures caused a higher and significant (p<0.001) reduction in growth rate. The major impact on size and volume sclerotia was produced by Ψ; followed by antioxidant mixtures. High AFB(1) levels were observed in response to the M1 applied at -7.1 MPa. Induction of the aflD gene was observed in response to the M1 treatment at -2.8, -7.1 and -9.9 MPa; but significant decreases of AFB(1) production and aflD transcripts were observed; when the fungus grew in the presence of the M2 treatment. These results showed that it is necessary to apply food-grade antioxidants into the peanut storage system at levels higher than 5 mM. This is an important tool to avoid sub-lethal antioxidant doses that can lead to fungal growth, increase resistance structures, and stimulate aflD gene expression and AFB(1) accumulation in this substrate.

Assessment of mycoflora and infestation of insects, vector of Aspergillus section Flavi, in stored peanut from Argentina.

The occurrence of spoilage fungi and Aspergillus section Flavi populations, the aflatoxins incidence, the role of insects as vectors of mycotoxin-producing fungi and the AFs-producing ability of the isolated species throughout the peanut (Arachis hypogaea L.) storage period were evaluated. Analyses of fungal populations from 95 peanut seed samples did not demonstrate significant differences between the incidences in each sampling period. Aspergillus section Flavi were isolated during all incubation periods. Cryptolestes spp. (Coleoptera: Cucujidae) were collected in August, September and October with 18, 16 and 28% of peanut samples contaminated, respectively. Insects isolated during August showed 69% of Aspergillus section Flavi contamination. A. flavus was the most frequently isolated (79%) from peanut seeds and from insect (59%). The greater levels of AFB1 were detected in September and October with a mean of 68.86 µg/kg and 69.12 µg/kg respectively. The highest proportion of A. flavus toxigenic strains (87.5%) was obtained in June. The presence of Aspergillus section Flavi and insect vectors of aflatoxigenic fungi presented a potential risk for aflatoxin production during the peanut storage period. Integrated management of fungi and insect vectors is in progress.

Impact of osmotic/matric stress and heat shock on environmental tolerance induction of bacterial biocontrol agents against Fusarium verticillioides.

Bacillus amyloliquefaciens and Microbacterium oleovorans reduced the Fusarium verticillioides count and significantly decreased fumonisin B(1) and B(2) levels in maize grains. The aim of this study was to determine the effect of water stress tolerance and heat shock survival upon cells of the biocontrol agents B. amyloliquefaciens and M. oleovorans. The a(w) of solid and liquid media and tryptic soy medium was modified to 0.99, 0.98, 0.97 and 0.96 by addition of ionic solute NaCl and non-ionic solutes such as glycerol and glucose. The non-ionic solute polyethylene glycol 600 (PEG 600) was used to modify matrically solid media. Bacterial incubation was at 30 °C. After 24, 48 and 72 h of incubation, samples from liquid media were spread-plate on nutrient agar medium and incubated for 24 h to determine the number of viable cells. The bacterial cells were harvested by centrifugation and heat treatment carried out in a water bath at 45 °C for 30 min. The viability of cells from different incubation times in liquid media showed statistically significant differences. Cells of B. amyloliquefaciens grown in liquid media amended with glycerol showed better tolerance at low a(w) and high survival under heat stress. These results could have important implications for optimizing and improving formulations.

Detection and quantification of Aspergillus section Flavi spp. in stored peanuts by real-time PCR of nor-1 gene, and effects of storage conditions on aflatoxin production.

Aspergillus flavus and A. parasiticus are the main species from section Flavi responsible for aflatoxin accumulation in stored peanuts. A real-time PCR (RT-PCR) system directed against the nor-1 gene of the aflatoxin biosynthetic pathway as target sequence was applied to monitor and quantify Aspergillus section Flavi population in peanuts. Kernels were conditioned at four water activity (a(W)) levels and stored during a 4-month period. The quantification of fungal genomic DNA in naturally contaminated peanut samples was performed using TaqMan fluorescent probe technology. Sensitivity tests demonstrated that DNA amounts accounting for a single conidium of A. parasiticus RCP08300 can be detected. A standard curve relating nor-1 copy numbers to colony forming units (cfu) was constructed. Counts of species of Aspergillus section Flavi from unknown samples obtained by molecular and conventional count (CC) methodologies were compared. A correlation between cfu data obtained by RT-PCR and CC methods was observed (r=0.613; p<0.0001); and the former always showed values higher by 0.5-1 log units. A decrease of fungal density was observed throughout the storage period, regardless of the quantification methodology applied. Total aflatoxin levels ranging from 1.1 to 200.4 ng/g were registered in peanuts conditioned at the higher a(W) values (0.94-0.84 a(W)). The RT-PCR assay developed appears to be a promising tool in the prediction of potential aflatoxigenic risk in stored peanuts, even in case of low-level infections, and suitable for rapid, automated and high throughput analysis.

Postharvest control of peanut Aspergillus section Flavi populations by a formulation of food-grade antioxidants.

The effect of a formulation containing butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl paraben (PP) on total mycoflora and Aspergillus section Flavi populations in natural and inoculated stored peanuts was evaluated. A survey of 480 peanut samples was carried out from July to December 2006. Two experimental units (silos 1 and 2) contained 200 kg of natural peanuts, while the other two (silos 3 and 4) had 200 kg of peanuts inoculated with Aspergillus flavus/A. parasiticus mixture (2 x 10(4) spores g(-1)). Silos 2 and 4 were treated with BHA-PP-BHT mixture (1802+1802+2204 microg g(-1)). Fungal counts were significantly affected (P<0.001) by Aspergillus section Flavi inoculum, tissue type, sampling period, antioxidant treatment and their interactions. Penicillium, Aspergillus and Fusarium spp. were the most common genera identified from both peanut tissues. Aspergillus flavus was the most frequently isolated species and there were significant differences (P<0.05) between its population in the control and treated peanuts. No aflatoxins were detected in any of the control or treated samples during storage. The development of natural peanut mycoflora and particularly Aspergillus section Flavi populations was inhibited by the ternary mixture of food-grade antioxidants.

Biological interactions to select biocontrol agents against toxigenic strains of Aspergillus flavus and Fusarium verticillioides from maize.

Biological control represent an alternative to the use of pesticides in crop protection. A key to progress in biological control to protect maize against Fusarium verticillioides and Aspergillus flavus maize pathogens are, to select in vitro, the best agent to be applied in the field. The aim of this study was to examine the antagonistic activity of bacterial and yeast isolates against F.verticillioides and A. flavus toxigenic strains. The first study showed the impact of Bacillus amyloliquefaciens BA-S13, Microbacterium oleovorans DMS 16091, Enterobacter hormomaechei EM-562T, and Kluyveromyces spp. L14 and L16 isolates on mycelial growth of two strains of A. flavus MPVPA 2092, 2094 and three strains of F. verticillioides MPVPA 285, 289, and 294 on 3% maize meal extract agar at different water activities (0.99, 0.97, 0.95, and 0.93). From this first assay antagonistics isolates M. oleovorans, B. amyloliquefaciens and Kluyveromyces sp. (L16) produced an increase of lag phase of growth and decreased a growth rate of all fungal strains. These isolates were selected for futher studies. In vitro non-rhizospheric maize soil (centrally and sprayed inoculated) and in vitro maize (ears apex and base inoculated) were treated with antagonistics and pathogenic strains alone in co-inoculated cultures. Bacillus amyloliquefaciens significantly reduced F. verticillioides and A. flavus count in maize soil inoculated centrally. Kluyveromyces sp. L16 reduced F. verticillioides and A. flavus count in maize soil inoculated by spray. Kluyveromyces sp. L16 was the most effective treatment limiting percent infections by F. verticillioides on the maize ears.