Molecular Identification and Mycotoxin Production by Alternaria Species Occurring on Durum Wheat, Showing Black Point Symptoms

Abstract

Black point is a fungal disease of wheat, mainly associated with mycotoxigenic Alternaria species. Affected wheat kernels are characterized by dark brown discolouration of the embryo region and reduction of grain quality. Potential risk is the possible accumulation of Alternaria mycotoxins, alternariol (AOH), alternariol-monomethyl ether (AME), tenuazonic acid (TA), and altenuene (ALT), provided by haemato-toxic, genotoxic, and mutagenic activities. One hundred and twenty durum wheat samples belonging to 30 different genotypes grown in Bologna and Modena areas, in Italy, showing black point symptoms, were analyzed for Alternaria species and their mycotoxin contamination. Alternariol was selected as an indicator of the capability of the Alternaria species to produce mycotoxin in vivo in field conditions. The data showed that Alternaria species occurred in 118 out of 120 wheat kernels samples, with the incidence of infected kernels ranging between 1% and 26%. Moreover, AOH was detected by using a HPLC with a diode array detector (LC-DAD) in 98 out of 120 samples with values ranging between 24 and 262 µg Kg^-1. Ninety-two Alternaria representative strains, previously identified morphologically, were identified at species/section level using gene sequencing, and therefore were analyzed for their mycotoxin profiles. Eighty-four strains, phylogenetically grouped in the Alternaria section, produced AOH, AME, and TA with values up to 8064, 14,341, and 3683 µg g^-1, respectively, analyzed by using a LC-DAD. On the other hand, eight Alternaria strains, included in Infectoriae Section, showed a very low or no capability to produce mycotoxins.

Keywords: altenuene; alternariol; alternariol-monomethyl ether; section Alternaria; section Infectoriae; species specific-primers; tenuazonic acid.

Figure 1

Phylogenetic tree generated by maximum parsimony method (bootstrap 1000 replicates) of combined allergen alt a1 (alt-a1), glyceraldeyde-3-phosphate dehydrogenase (gpd), and translation elongation factor 1-α (tef) gene sequences of 92 Alternaria strains.

Figure 2

Deletion of 55 nt in gpd sequence of Alternaria strains belonging to Infectoriae section. Ainf29Fw primer (5′-CGTCTTCCGCAATGCTATCG3-3′), designed on deletion site, was able to discriminate among Alternaria and Infectoriae Sections.