Morphological and Molecular Characterization of Some Alternaria Species Isolated from Tomato Fruits Concerning Mycotoxin Production and Polyketide Synthase Genes

Abstract

Tomatoes (Lycopersicon esculentum) are one of the main crops grown in Egypt. The fungal black spot illness of fruits is usually associated with the secretion of mycotoxin by Alternaria toxigenic species. Twenty Alternaria isolates were isolated from infected tomatoes fruits by baiting technique, morphologically identified to species level, and confirmed using Internal Transcribed Spacer (ITS) gene sequencing. ITS gene sequencing of fragments obtained 547, 547, 542, 554, and 547 bp for A. alternata, A. brassicicola, A. citri, A. radicina, and A. tenuissima, respectively. Alternaria species were investigated for mycotoxin production using the high-performance liquid chromatography (HPLC) technique. The data from the HPLC analysis showed that the mycotoxins were determined in four out of five Alternaria species, with the incidence ranging from 0.89-9.85 µg/mL of fungal extract at different retention times. Alternaria alternata was the most active species and produced three types of toxins. Polyketide synthase genes (pksH and pksJ) which are involved in the Alternaria toxin's biosynthesis were also amplified from the DNA of Alternaria species.

Keywords: Alternaria; HPLC; internal transcribed spacer; mycotoxins; pksH & pksJ genes.

Figure 1

Alternaria species isolated from infected tomato fruits.

Figure 1

Alternaria species isolated from infected tomato fruits.

Figure 2

Agarose gel electrophoresis of PCR amplification of DNA products, PCR amplification of DNA products (~600 bp) using ITS1/ITS4 primer pair. L—ladder (100–1000 bp); P—positive control consists of a segment of DNA of known size (the same size as the target amplicon, shows that the primers have attached to the DNA strand); N—negative control: a sample without DNA, but contains all essential components of the amplification reaction show if contamination of the PCR experiment with foreign DNA has occurred; 2—A. alternata; 5—A. citri; 10—A. brassicicola; 14—A. radicina; 20—A. tenuissima (A) and phylogenetic tree generated from Alternaria species used in this study based on datasets of ITS rDNA gene sequences, the similarity of 97–98% among species (B).

Figure 3

Phylogenetic tree generated from Alternaria species and its related species/isolates based on datasets of ITS rDNA gene sequences. There is similarity of 99–100% among species. Alternaria isolates with a star were recovered from tomato fruits in this study.

Figure 4

Agarose gel electrophoresis of PCR amplification of pksH (290 bp) and pksJ (260 bp) genes from Alternaria species: L—ladder (100–1000 bp); P—positive control consists of a segment of DNA of known size (the same size as the target amplicon, shows that the primers have attached to the DNA strand); N—negative control: a sample without DNA, but contains all essential components of the amplification reaction shows if contamination of the PCR experiment with foreign DNA has occurred; 2—A. alternata; 5—A. citri; 10—A. brassicicola; 14—A. radicina; 20—A. tenuissima.

Figure 5

HPLC analysis of Alternaria culture extracts for mycotoxin detection: A. alternata (A); A. citri (B); A. brassicicola (C); A. radicina (D); A. tenuissima (E); toxin standard (F); AOH, AME, ALT, TA, respectively.