Molecular interactions of arbuscular mycorrhizal fungi with mycotoxin-producing fungi and their role in plant defense responses

Fusarium trichothecenes are a large group of sesquiterpenes that are inhibitors of eukaryotic protein synthesis. They cause health problems for humans and animals that consume fungus-infected agricultural products. In addition some of Fusarium trichothecenes are virulence factors of plant pathogenesis. In this study, sixteen Fusarium strains were isolated and characterized from naturally infected potato plants. Pathogenicity tests were carried out to evaluate the virulence of these isolates on potato plants and their trichothecene production capacity. We chose F. sambucinum strain T5 as a model for this study because it was the most aggressive strain when tested on potato plants. It induces a rapid wilting and yellowing resulting in plant death. This strain produced 4,15-diacetoxyscirpenol (4,15-DAS) when grown in liquid culture. We amplified and characterized five trichothecene genes (TRI5, TRI4, TRI3, TRI11, and TRI101) involved in the production of 4,15-DAS. Nucleotide BLAST search showed 98% and 97% sequence identity with trichothecene biosynthetic genes of F. sporotrichioides and Gibberella zeae, respectively. We used F. sambucinum to determine if trichothecene gene expression was affected by the symbiotic arbuscular mycorrhizal fungus (AMF) Glomus irregulare. We found that the growth of F. sambucinum was significantly reduced in the presence of G. irregulare isolate DAOM-197198 compared with controls that consisted of carrot roots without G. irregulare or F. sambucinum alone. Furthermore, inhibition of the growth F. sambucinum was associated with morphological changes, which were observed when G. irregulare hyphae reached F. sambucinum mycelium, suggesting that G. irregulare may produce compounds that interfere with the growth of F. sambucinum. Using real-time qRT-PCR assays, we assessed the relative expression of trichothecene genes of F. sambucinum confronted or not with G. irregulare. When G. irregulare was confronted with F. sambucinum, TRI5 and TRI6 genes were up-regulated, while TRI4, TRI13 and TRI101 were down-regulated. We therefore used GC-MS analysis to determine whether G. irregulare affects trichothecene production by F. sambucinum. We found that the production of 4,15-DAS trichothecene was significantly reduced in the presence of G. irregulare compared with controls that consisted of carrot roots without G. irregulare or F. sambucinum alone. Interestingly, 4,15-DAS pattern was reduced to 39 μg/ml GYEP medium by G. irregulare compared to 144 μg/ml GYEP with F. sambucinum grown with carrot roots or F. sambucinum alone respectively. We tested the AMF capacity to induce defense responses of potato plants following infection with F. sambucinum. The response of AMF-colonized potatoes to F. sambucinum was investigated by tracking the expression of genes homologous with pathogenesis-related proteins chitinase class II (ChtA3), 1,3-β-glucanase (gluB), peroxidase (CEVI16), osmotin-like protein (OSM-8e) and pathogenesis-related protein (PR-1). We found that the AMF treatment up-regulated the expression of all defense genes in roots at 72 hours post-infection (hpi) with F. sambucinum. We also found that a decrease provoked by F. sambucinum in gluB and CEVI16 expression in shoots could be blocked by AMF treatment. Overall, a differential regulation of PR homologues genes in shoots indicates that AMF are a systemic bio-inducer and their effects could extend into non-infected parts. In conclusion, this study provides new insight into on the interactions between plants and microbes, in particular the effects of AMF on the growth and the reduction of mycotoxins in Fusarium. It also shows that AMF are able to reduce the disease severity in plants by stimulating their defense. The data presented provide new opportunities for bio-control against mycotoxin-producing pathogens in plants.

Keyphrases: Fusarium sambucinum, 4,15-diacetoxyscirpenol (4,15-DAS), qRT-PCR, Mycotoxins, Fusarium sambucinum, Trichothecenes cluster genes, 4,15-diactoxycscirpenol (4,15-DAS), qRT-PCR, Gene expression, Arbuscular mycorrhizal fungi, defense related genes