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.

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.

Application of essential oils in maize grain: impact on Aspergillus section Flavi growth parameters and aflatoxin accumulation.

The antifungal activity of Pimpinella anisum L. (anise), Pëumus boldus Mol (boldus), Hedeoma multiflora Benth (mountain thyme), Syzygium aromaticum L. (clove), and Lippia turbinate var. integrifolia (griseb) (poleo) essential oils (EOs) against Aspergillus section Flavi was evaluated in sterile maize grain under different water activity (a(w)) condition (0.982, 0.955, and 0.90). The effect of EOs added to maize grains on growth rate, lag phase, and aflatoxin B(1) (AFB(1)) accumulation of Aspergillus section Flavi were evaluated at different water activity conditions. The five EOs analyzed have been shown to influence lag phase and growth rate. Their efficacy depended mainly on the essential oil concentrations and substrate water activity conditions. All EOs showed significant impact on AFB(1) accumulation. This effect was closely dependent on the water activity, concentration, and incubation periods. Important reduction of AFB(1) accumulation was observed in the majority of EO treatments at 11 days of incubation. Boldus, poleo, and mountain thyme EO completely inhibited AFB(1) at 2000 and 3000 microg g(-1). Inhibition of AFB(1) accumulation was also observed when aflatoxigenic isolates grew with different concentration of EOs during 35 days.

Antiaflatoxigenic property of food grade antioxidants under different conditions of water activity in peanut grains.

Analytical grade (AG) and industrial grade (IG) of three-food grade antioxidants butylated hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and propyl paraben (PP) were analyzed to prove their fungitoxic effect on Aspergillus section Flavi strains. The effect of interactions among 10 antioxidant treatments at water activity levels (0.982, 0.955, 0.937 a(W)) for 11 and 35 days of incubation and at 25 degrees C in peanut grains on mycelial growth (CFU g(-1)) and aflatoxin B(1) (AFB(1)) accumulation were evaluated. Both antioxidant grade treatments had a significant effect (P<0.001) on fungal count. All antioxidant treatments showed the highest effectiveness on control of growth of peanut aflatoxigenic strains at 0.937 a(W) and at 11 days of incubation. Overall, AG and IG binary mixtures M3 (20+10 mM), M4 (20+20 mM) and ternary mixtures M5 (10+10+10 mM), M6 (10+20+10 mM), M7 (20+10+10 mM) and M8 (20+20+10 mM) were the treatments most effective at inhibiting growth of Aspergillus section Flavi strains. Industrial grade BHA 10 and 20 mM, binary mixtures M1 (10+10 mM), M2 (10+20 mM), M3 (20+10 mM), M4 (20+20 mM) and ternary mixtures M5 (10+10+10 mM), M6 (10+20+10 mM), M7 (20+10+10 mM) and M8 (20+20+10 mM) completely inhibited AFB(1) production. The studied results suggest that IG antioxidant mixtures have potential for controlling growth of these mycotoxigenic species and prevent aflatoxin accumulation at the peanut storage system.

Control of Aspergillus growth and aflatoxin production using natural maize phytochemicals under different conditions of water activity.

The effects of the natural phytochemicals trans-cinnamic acid (CA) and ferulic acid (FA) alone at concentrations of 1-25 mM and in 16 combinations (M: mixtures) on growth and aflatoxin B(1) production by Aspergillus flavus Link and A. parasiticus Speare were evaluated. Studies on growth rate and aflatoxin B(1) production were carried out in vitro in relation to a water activity a(w) of 0.999, 0.971, 0.955 and 0.937. Overall, CA at concentrations of 10 and 20 mM and FA-CA mixtures M3 (20 + 5 mM respectively), M8 (25 + 5 mM), M9 (1 + 10 mM), M10 (10 + 10 mM), M11 (20 + 10 mM), M12 (25 + 10 mM), M13 (1 + 20 mM), M14 (10 + 20 mM), M15 (20 + 20 mM) and M16 (25 + 20 mM) were the treatments most effective at inhibiting growth of the four species assayed. All strains were much more sensitive to all natural phytochemicals tested on growth rate at a(w) = 0.937. CA and the FA-CA mixtures M1 (1 + 1 mM respectively), M4 (25 + 1 mM), M5 (1 + 5 mM), M6 (10 + 1 mM), M7 (20 + 1 mM), M8 (25 + 5 mM), M9 (1 + 10 mM), M10 (10 + 10 mM), M11 (20 + 10 mM), M12 (25 + 10 mM), M13 (1 + 20 mM), M14 (10 + 20 mM), M15 (20 + 20 mM) and M16 (25 + 20 mM) completely inhibited aflatoxin B(1) production by all strains at a(w) = 0.999, 0.971, 0.955 and 0.937. Decreased aflatoxin B(1) levels in comparison with the control were observed with FA at 1, 10, 20 and 25 mM with the strains RCM89, RCM108 and RCM38 at a(w) = 0.971, 0.955 and 0.999 respectively. The data show that CA and FA can be considered as effective fungitoxicants for A. flavus and A. parasiticus in in vitro assay. The information obtained is part of an ongoing study to determine their application at the storage level.

Influence of Bacillus spp. isolated from maize agroecosystem on growth and aflatoxin B(1) production by Aspergillus section Flavi.

A total of 59 bacteria of the Bacillus genus were isolated from different components of a maize agroecosystem and their antifungal activity against Aspergillus section Flavi was evaluated. Thirty-three and 46% of these bacteria were able to inhibit Aspergillus flavus Link and A. parasiticus Speare respectively at water activity (a(w)) 0.982; however, when a(w) was 0.955, these percentages were decreased and only three isolates were able to inhibit Aspergillus section Flavi. The majority of bacilli acted as contact antagonists, while a small number of isolates were able to form inhibition zones. In maize meal extract agar, Aspergillus section Flavi growth rate and aflatoxin B(1) (AFB(1)) production were significantly reduced when these strains were paired at a(w) 0.982 with bacilli at all inoculum levels studied. However, two bacilli isolated were able to reduce growth rate and aflatoxin production when a(w) was 0.955. Lag phase increase followed the same general pattern as growth rate reduction. When Aspergillus section Flavi was grown in sterile maize in the presence of three Bacillus strains at a(w) 0.982, the reduction in count (colony-forming units (cfu) g(-1) maize) was less than 30%, except when Aspergillus section Flavi grew with Bacillus amyloliquefaciens UNRCLR. However, levels of detectable AFB(1) were significantly reduced in these interactions at a(w) 0.982.

Rhizobacteria and their potential to control Fusarium verticillioides: effect of maize bacterisation and inoculum density.

Fusarium verticillioides is the most important seed transmitted pathogen that infects maize. It produces fumonisins, toxins that have potential toxicity for humans and animals. Control of F. verticillioides colonisation and systemic contamination of maize has become a priority area in food safety research. The aims of this research were (1) to characterise the maize endorhizosphere and rhizoplane inhabitant bacteria and Fusarium spp., (2) to select bacterial strains with impact on F. verticillioides growth and fumonisin B1 production in vitro, (3) to examine the effects of bacterial inoculum levels on F. verticillioides root colonisation under greenhouse conditions. Arthrobacter spp. and Azotobacter spp. were the predominant genera isolated from maize endorhizosphere and rhizoplane at the first sampling period, whilst F. verticillioides strains showed the greatest counts at the same isolation period. All F. verticillioides strains were able to produce fumonisin B1 in maize cultures. Arthrobacter globiformis RC5 and Azotobacter armeniacus RC2, used alone or in a mix, demonstrated important effects on F. verticillioides growth and fumonisin B1 suppression in vitro. Only Azotobacter armeniacus RC2 significantly reduced the F. verticillioides root colonisation at 10(6) and 10(7) CFU g(-1) levels under greenhouse conditions.

Alternaria Mycotoxins in Sunflower Seeds: Incidence and Distribution of the Toxins in Oil and Meal.

A survey of 150 sunflower-seed samples was carried out to evaluate the contamination from infection with Alternaria alternata with alternariol (AOH), alternariol monomethyl ether (AME) and tenuazonic acid (TA). A high percentage of the samples was contaminated with AOH (85%), AME, (47%), and TA (65%). The average levels detected were 187 µg/kg for AOH, 194 µg/kg for AME, and 6,692, µg/kg for TA. When sunflower seeds fermented by Alternaria alternata were processed under laboratory conditions to obtain the oil and meal, different distributions of Alternaria toxins between the oil and the meal were observed: whereas AOH, AME, and TA were detected in the meal, only AME and TA were detected in the oil, and the latter in a low percentage.