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Biocontrol of Aflatoxin Contamination Using Atoxigenic Strains from Almond and Pistachio Orchards

Periodic Reporting for period 2 - BIOCONTROL-A (Biocontrol of Aflatoxin Contamination Using Atoxigenic Strains from Almond and Pistachio Orchards)

Reporting period: 2018-06-01 to 2019-05-31

Aflatoxin contamination of essential crops caused by Aspergillus flavus and closely-related fungal species frequently occur in warm agricultural areas. Aflatoxin affects health, trade, and development sectors of affected nations. Because these fungi are ordinary soil residents of almond and pistachio orchards, and their spores are spread using the air currents, these nuts are a source of human exposure to aflatoxin
According to data provided by the Rapid Alert System for Food and Feed (RASFF, 2019), aflatoxins are currently responsible for most mycotoxins alerts reported in the European Union (Figure 1).
Application of atoxigenic (not producing aflatoxins) strains of A. flavus has successfully reduced crop aflatoxin-contamination in USA and Africa, and recently in Italy. In California, the atoxigenic strains AF36 is applied by the farmers as sorghum seeds colonized by this atoxigenic strain using the registered compound AF36 Prevail®.
This biological control strategy uses endemic atoxigenic A. flavus strains, considered best adapted, to displace the AF-producing fungi. Unfortunately, EU nut farmers do not have any benefit of this type of biological control technology since there are no registered atoxigenic strains in these crops.
The aim of the European phase (in Spain and Italy) of the present project was to identify native biological control strains and develop mechanistic models for understanding the biological control process.
The expected results will have a positive effect on improving food safety and the environment and securing economic benefits to EU farmers and agri-food industries.
Objective 4. Study the incidence of aflatoxin in almond and pistachio orchards and select atoxigenic A. flavus strains in Spain.

We have collected around 100 isolates from different Spanish Nut orchards. Firstly, we developed protocol methods for isolation of these species from Spanish soils using AFAPA and Si. Subsequently, the Aspergillus strains were identified according to their morphological characteristics (mainly type of spores and conidiophore). Those strains could not be unequivocally identified according to their morphological characteristics were identified according to their molecular profile using three DNA regions, ITS, Calmodulin, Bt (Varga et al., 2011; Studies n Mycology 69:57-80). Thirty isolates were characterized according to their ability to produce aflatoxin using Coconut Medium made with powdered coconut. Finally, the aflatoxin produced for the toxic isolates were quantified using an HPLC (Doster et. al. 2014: Plant Dis. 98:948-956). In addition, to identify risk factor associated with the aflatoxin contamination in nut crops in Spain, the susceptibility of different Spanish almond cultivars has been evaluated by artificial inoculations (Moral et al., Phytopathology 107(12): 75). According to our preliminary results, native atoxigenic strains of A. flavus have been isolated for future evaluations. Also, we have identified some Spanish almond cultivars moderately resistant to Aspergillus (then, aflatoxin contamination).

Objective 5. Develop predictive models for aflatoxin contamination in almond and pistachio

The entire set data obtained during the BIOCONTROL-A project and data coming from Michailides’ group have been used for: developing a sensitivity analysis and a mechanistic model to study the competence between toxin and atoxic isolates (Battilani et al. 2012. EFSA report 009-00812. Rossi et al. 2010. in: The Challenge and Use of Heterogeneity, 241-248.). This objective was conducted during a 1-month stay of Dr. Moral in the USCU (Rossi´s Lab). Currently, the sensitivity model is doing validated. In the case of the mechanistic model is still under develop. Interstate, and according to the senility model, in common seasons with low-common percentage of pistachio fruits showing Early Split (ES) or low population of pests in California, the aflatoxin contamination risk is very low, making unnecessary the treatments with biological control agents.
Aflatoxin contamination of economically important crops occurs more frequently in tropical and subtropical regions (Klich 2007; JECFA 2018). Susceptible crops include maize, groundnut, cottonseed, tree nuts, figs, chilies, among others (Cotty et al. 1994; Bandyopadhyay et al. 2007; Ortega-Beltran et al. 2018). Aflatoxins are synthesized alone or in conjunction with other mycotoxins by a diversity of species within Aspergillus sect. Flavi. Cultural practices and fungicide sprays have not been effective in controlling AF contamination. Naturally-occurring atoxigenic strains of A. flavus have been used successfully to reduce AF contamination of crops in the USA, Africa (Dorner. 2009. J Food Protect 72:801-804), and, recently, in Italy (Mauro et al. 2018. Toxins. DOI: 10.3390/toxins10010030).

All the Aspergillus Flavi isolates from Spanish nut soils belong to the species A. flavus. The Spanish population of this species is composed of toxic and nontoxic strains, which could be used as potential biocontrol agents. Furthermore, A. parasiticus population, which is characterized because all their isolates are toxic, is apparently lower than in USA soils.

The results of the BIOCONTROL-A project during its return phase have helped to identified atoxigenic strains of A. flavus.
European alerts about mycotoxin contamination