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BIOCONTROL-A Report Summary

Project ID: 658579
Funded under: H2020-EU.1.3.2.

Periodic Reporting for period 1 - BIOCONTROL-A (Biocontrol of Aflatoxin Contamination Using Atoxigenic Strains from Almond and Pistachio Orchards)

Reporting period: 2016-09-01 to 2018-05-31

Summary of the context and overall objectives of the project

Aflatoxins (AF) are potent carcinogens produced by Aspergillus species. Because these fungi are common soil residents of almond and pistachio orchards, and their spores are spread by means of the air currents, these nuts are a source of human exposure to AF. In 2016, the AFs [levels: 10 µg/Kg total AFs (B1, B2, G1, and G2) for fresh consumption] came as the first mentioned hazard contamination according to the number of notification by country (Rapid Alert System for Food and Feed, RASFF).

Application of atoxigenic (not producing AFs) strains of A. flavus has successfully reduced crop AF-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 registred 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 the benefit of this type of biological control technology since there are not registered atoxigenic strains in these crops. The aim of the outgoing phase of the present project is to improve the biological control of AFs.

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.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

Obj. 1. Study the displacement of the aflatoxin-producing A. flavus strains by the atoxigenic AF36 in almond orchards
We initiated a continuous monitoring of the AF36 levels in commercial orchards in 2012, and by 2016/2017 the percentage of A. flavus isolates belonging to the atoxigenic strain AF36 was 50% in orchards continuously treated while nearby orchards that were never treated had 18.9%. The density of A. flavus/A. parasiticus in the soil was not significantly different for the two application rates (5 vs 10 kg / ha), and no significant differences were observed in the species in the soil between orchards treated with different rates kg/ha. Because the latter results, together with some studies, which showed the low impact of the biological control agents in the honey bees, in March 2017 United States Environmental Protection Agency (USEPA) approved the registration of AF36 Prevail for use in almond orchards in USA (Ortega-Beltran & Moral et al. 2018. PlosOne.
Obj. 2. Improve the substrate and application methods of atoxigenic A. flavus strains
The initial objective was for comparing sorghum-AF36 product versus wheat-AF36 product. Applying the seed-coated sorghum-AF36 product and the seed-coated wheat-AF36 product both resulted in substantial increases (none differences) in the atoxigenic strain AF36 in the treated areas. The results demonstrated that the sorghum-AF36 product would be an adequate alternative to the wheat-AF36 product. When different sorghum cultivars were evaluated, AF36 sporulated more on low phenol-containing cultivars than in all other cultivars tested, including the wheat formulation.

In this project, we have reported additional VCGs associated with almond and pistachio crops. Representative isolates of 12 atoxigenic VCGs significantly (P < 0.001) reduced (> 80%) aflatoxin accumulation in almond and pistachio kernels when challenged with highly toxigenic isolates of A. flavus or A. parasiticus. Application of several biological control strains would favor long-term atoxigenic A. flavus communities across the nut-growing areas and this would result in health benefits to consumers (Ortega-Beltran & Moral et al., 2018. Plant Dis. In Press). Likewise, Dr. Camiletti identified several Argentinean atoxigenic A. flavus strains from maize in Michailides´ Lab under J. Moral supervision (Camiletti & Moral et al., 2018. Phytopathology 108:808-818).
Objective 4. Study the incidence of AF in almond and pistachio orchards and select atoxigenic A. flavus strains Spain
However, this objective is going to be guided during my current return phase. We have studied the studied the resistance/susceptibility of Spanish almond cultivars among others (Moral et al. 2017. Phytopathology 107:S5.75).

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The aflatoxins are potent liver carcinogens and outbreaks of acute aflatoxins can result in the death of several people (Lewis et al. 2005. Environ Health Persp 113:1763-1767). 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 USA, Africa (Dorner. 2009. J Food Protect 72:801-804), and, recently, in Italy (Mauro et al. 2018. Toxins. DOI: 10.3390/toxins10010030).
Here, we have shown that application of AF36 atoxigenic strain has low impact on honey bees and, also, that allows maintaining a high density of this atoxic strains across the time (Ortega-Beltrán & Moral et al. 2018. PlosOne. Likewise, other atoxigenic and native strains of A. flavus have been identified looking for increasing the diversity of biological control strains (Ortega-Beltran & Moral et al., 2018. Plant Dis. In Press). Finally, we are paying attention to cyclopiazonic acid (CPA), a less-known mycotoxin also produced by Aspergillus species (Camiletti & Moral et al., 2018. Phytopathology 108:808-818). Also, during the outgoing phase of this project at UC-Davis, Dr. Moral has co-supervised several students from different countries such as Dr. C. Agustí (Postdoctoral researcher), Ms. T. García (Ph.D. Student), Mr. Boris Camiletti (Ph.D. Student), Mr. Chirag Vazirani (Master Student), Mr. K. Tomari (Master Student), Mr. A. Papagelis (Undergraduate Student), and Ms. Alexi Jackson (Undergraduate Student).
The results of the BIOCONTROL-A project during its first phase have helped the registration of AF36 Prevail® for using in Californian almond and fig orchards. Although this participation has been limited considering that the experiments were initiated in 2001 and the registration was on March 2017 ( Moreover, Registered Intellectual Property Rights does not affect this project since Dr. Cotty (United States Patent No. 5, 171, 86-December 15, 1992 and United States Patent No. 5, 294, 442-March 15, 1994) obtained the patent for the use of the non-toxigenic strain AF36 of A. flavus as biological control organism.
During the following research phase of our BIOCONTROL-A project, we are going to apply the knowledge and technical abilities, which have been acquired by the Dr. Moral, on understanding the aflatoxin contamination risks in Southern Spain, and on the identification of endemic biological control strains (Ortega-Beltran & Moral et al., 2018. Plant Dis. In Press). For publishing, we will prioritize their publication in Open access journal, or through posting them in freely availably preprint servers such as ResearchGate (

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