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.

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.

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.

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.