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Significant variations in the macromorphological characteristics of the 40 analyzed E. sorghinum isolates were observed, as expected considering the high intraspecific variation presented by this group of fungi[33]. This fact allowed us to differentiate the isolates into three specifically defined groups (A, B and C) based, mainly, on their growth rate and their ability to produce pigments and exudates (Fig. 1). All the characteristics described below were observed after 7 d of incubation in MEA medium. Group A included 12 isolates with smaller diameter colonies, visibly furrowed, mycelium with a light centre that becomes darker towards the periphery and pink edges; intense pink reverse, especially in the centre, the intensity of the colour decreases towards the periphery of the colony, with the youngest mycelium being whitish. Some of these isolates presented transparent exudates. On the other hand, 21 isolates are classified within group B and the colonies, in this case, had an intermediate diameter, floccose but not scarce with mycelium from pink in the centre to greyish green towards the periphery, culminating in whitish colour; reverse of homogeneous intense brown to almost black colour, with reddish pigment that extends to the culture medium. Finally, group C was represented by seven isolates whose colonies showed the greatest development over time, presenting soft grooves, a light brown centre that turns salmon, with white edges; clear back, light salmon centre that turns whitish towards the periphery.
Figure 1.
Macroscopic characteristics of E. sorghinum: colony on MEA medium after 7 incubation days (front and reverse). Representatives of morphological groups A, B and C.
The members of the B group covered the whole Petri dish, in contradistinction to the members of the other two groups whose colonies reached a maximum diameter between 60−75 mm.
The macromorphology observed in the colonies grown in OA was similar for all the analyzed isolates, showing no differences between the morphological groups described. The colony reached a diameter between 50−90 mm, with regular edges, and olive-green conidia. The colonies showed a compact and felty texture, while on the reverse a brown pigmentation was observed.
All the studied isolates showed typical micromorphological characteristics of this species: brown globose pycnidia with a straight neck, of 69 to 11.5 × 44.5 to 90 µm in size (n = 50); multicellular hyaline to brown chlamydospores, massively produced with measurements ranging between 3 and 50 μm, and mostly ovoid conidia of 3.5 to 5.5 × 1.8 to 3 µm (n = 50) (Fig. 2).
Figure 2.
Micromorphological structures of E. sorghinum. (a) hyaline/brown dictyochlamydospores, and (b) Hyaline/brown glabrous pycnidia. Scale bars = 10 μm.
Tenuazonic acid production
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A considerable variability in mycotoxin production was also observed as it happened with the morphological diversity. Regarding the toxigenic capacity of E. sorghinum, the results suggest a toxicological risk for animals exposed to tenuazonic acid (TeA) through the consumption of feed contaminated with this producer species. This assertion is based on the correlation between the sorghum samples contaminated with TeA and the origin of the isolates that turned out to be TEA-producers isolates. Sixty-five percent of the analyzed isolates (n = 40) were producers of TeA with levels that ranged from 112 to 47,237 μg·kg−1. Table 1 shows the TeA concentration by the 26 TeA-producing E. sorghinum isolates.
Table 1. Tenuazonic acid (TeA) concentration produced by assayed E. sorghinum isolates and their corresponding morphological group.
Morphological group Isolates TeA concentration (μg·kg−1) A LMCIN-2.1 13,846.39 LMCIN-5.3 7,556.21 LMCIN-5.7 112.45 LMCIN-5.11 11,948.50 LMCIN-7.1 28,000.83 LMCIN-8.1 2,001.46 LMCIN-9.6 1,679.33 LMCIN-9.11 5,129.46 LMCIN-11.1 548.78 LMCIN-12.4 3,870.20 LMCIN-18.4 721.33 B LMCIN-1.12 5,890.30 LMCIN-3.2 16,395.00 LMCIN-3.11 1,289.45 LMCIN-7.2 3,058.24 LMCIN-7.3 389.49 LMCIN-9.3 7,790.25 LMCIN-9.10 5,900.17 LMCIN-11.7 18,374.58 LMCIN-13.5 47,237.28 LMCIN-15.3 6,720.12 LMCIN-16.4 3,245.55 C LMCIN-6.9 2,190.36 LMCIN-12.8 4,069.33 LMCIN-18.7 16,280.38 LMCIN-19.2 8,130.35 Molecular characterization
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The phylogenetic analysis of the ITS and TUB2 sequences separately and the morphological characteristics finally allowed us to identify the analyzed strains as E. sorghinum. The analysis by Blast database could identify the majority of the isolates as E. sorghinum, whose e-values and percentage of identity were of 0.0 and 100%, respectively. Except for the isolate LMCIN-18.4 (e-value 0.0 and percentage of identity 100%) that was identified as Phoma sp.
Most of the isolates showed 100% similarity in the analyzed sequences, therefore not all of them were included in the phylogenetic analysis, since the objective of the work was to verify the morphological identification and to determine the degree of similarity with those reported strains.
Table 2 shows the access number of GenBank of the nucleotide sequences of the assayed isolates in this study and the number access of the sequences used by the phylogenetic analysis.
Table 2. Reference sequences selected according to the taxonomic closeness and downloaded from GenBank to construct the phylogenetic tree.
Species Isolate Country GenBank accession ITS TUB2 E. sorghinum LMCIN-1.12 Argentine OQ971382 OR125009 E. sorghinum LMCIN-1.12 Argentine OQ971382 OR125010 E. sorghinum LMCIN-5.11 Argentine OQ971384 OR125011 E. sorghinum LMCIN-6.9 Argentine OQ971385 OR125012 E. sorghinum LMCIN-9.6 Argentine OQ971386 OR125013 E. sorghinum LMCIN-9.11 Argentine OQ971387 OR125014 E. sorghinum LMCIN-11.7 Argentine OQ971388 OR125015 E. sorghinum LMCIN-12.8 Argentine OQ971389 OR125016 E. sorghinum LMCIN-18.4 Argentine OQ971390 OR125017 E. sorghinum CBS 179.80 Puerto Rico FJ427067 FJ427173 E. sorghinum CBS 627.68 France FJ427072 FJ427178 E. sorghinum LC 4860 China KY742116 KY742358 E. viticis LC 5126 China KY742118 KY742360 E. camelliae LC 4858 China KY742091 KY742333 E. latusicollum LC 5158 China KY742101 KY742343 E. pimprinum CBS 246.60 India FJ427049 FJ427159 E. longiostiolatum CBS 886.95 Papua New Guinea FJ427074 FJ427180 Leptosphaeria doliolum CBS 505.75 Netherlands JF740205 JF740144 Figure 3 shows the phylogenetic dendrogram constructed starting the combined ITS + TUB2. Associations between macro morphological variation, toxicogenic capacity and the phylogenetic results were found. In this way, LMCIN-9.11, LMCIN-9.6 and LMCIN-5.11 isolates were grouped in a cluster presenting all these isolates the morphology described within group A; on the other hand, isolates representatives of morphological group B were grouped into the following cluster: LMCIN-1.12, LMCIN-3.11 and LMCIN-11.7 isolates. It is highlight that all the isolates mentioned above are mycotoxin-producing isolates while the two non-mycotoxin-producing isolates included in the molecular analysis (LMCIN-6.9 and LMCIN-12.8) are located in the same subnode, however, in this case there was no correlation with the macromorphological characteristics since they belong to different morphological groups.
Figure 3.
Phylogenetic tree of the Epicoccum isolates obtained from sorghum samples, derived from sequences of the ITS and β-tubulin region of the nuclear ribosomal DNA.
Other results of the taxonomic search for isolates: LMCIN-1.12, LMCIN-3.11, LMCIN-6.9, LMCIN-11.7, LMCIN-12.8, were relationated to Epicoccum latusicollum with similar values confidence to obtained above.
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All data generated or analyzed during this study are included in this published article.
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About this article
Cite this article
Hipperdinger ML, Colman DI, Gortari MC, Pereyra CM, Astoreca AL. 2024. Characterization of Epicoccum isolates obtained from Argentinean sorghum grain samples. Studies in Fungi 9: e003 doi: 10.48130/sif-0024-0004
Characterization of Epicoccum isolates obtained from Argentinean sorghum grain samples
- Received: 04 November 2023
- Revised: 13 March 2024
- Accepted: 21 March 2024
- Published online: 16 April 2024
Abstract: Sorghum has numerous agronomic advantages, a great economic importance in food production and various industrial applications. Its consumption has increased in the last ten years and probably its importance may even increase in the future, considering its relationship with global warming since this plant is less demanding with water. However, its productivity is affected by various fungal diseases with the production of mycotoxins that cause great economic losses. Alternaria, Epicoccum and Pyricularia genera are the main fungal contaminants in sorghum grains, and recognized producers of tenuazonic acid, a mycotoxin previously found in assayed sorghum samples in the Mycology and Mycotoxicology laboratory belonging to the Center for Research and Development in Industrial Fermentations. Fungal isolates obtained from these sorghum grains from the National Institute of Agricultural Technology (INTA, Manfredi, Córdoba, Argentina) were characterized using a polyphasic approach based on morphological and genetic characteristics and in the ability to produce mycotoxins. Morphological analysis suggested the identity of Epicoccum sorghinum, which was later confirmed by molecular analysis. The ability of these isolates to produce tenuazonic acid was evaluated and it was determined that 65% of the studied isolates produced tenuazonic acid at variable levels. This is the first study that provides a molecular approach to E. sorghinum isolates in Argentina and clearly confirms the wide genetic and phenotypic variability previously reported for this species in other countries. The presence of these tenuazonic acid-producing isolates in sorghum grains represent an economic and health problem for Argentina that it is considered one of the main exporters worldwide.
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Key words:
- Cereals /
- Food safety /
- Fungi /
- Phenotypic and molecular analysis /
- Tenuazonic acid