María Alejandra Passone

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.

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.

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.

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.

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 β-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.

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.

Effects of sub-lethal food grade antioxidant doses and environmental stressors on growth, sclerotia, aflatoxins and aflD (nor-1) expression by Aspergillus parasiticus RCP08300

The aim of the work was to examine the effects of sub-lethal doses of butylated hydroxyanisole (BHA) and butylated hydroxytoluene (BHT) (1+1 mmol/l and 5+5 mmol/l), water activities (0.98, 0.95, 0.93, 0.89 aW) and temperatures (28, 20 °C) on growth, sclerotial characteristics, aflatoxin accumulation and aflD (=nor-1) transcript level by Aspergillus parasiticus RCP08300 on peanut based medium. Growth rate and aflatoxin production were inhibited by BHA-BHT mixture (1+1 mmol/l), regardless of environmental factor assayed. Although sclerotia number and aflD expression were stimulated by this treatment, sclerotia dry weight and volume were reduced by 62.3 and 31.2%, respectively. In contrast, when the fungus grew in presence of the higher dose of BHA-BHT mixture none or very low aflatoxin accumulation and aflD expression occurred. Similarly, A. parasiticus growth has been highly influenced by BHA-BHT (5+5 mmol/l) and interacting stress factors. Data show that sub-lethal antioxidant doses significantly reduced ...

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.Graphical Abstract