Adaptation of the quantitative PCR method for the detection of the main representatives of cereal grain mycobiota

Abstract

The content of fungal DNA and mycotoxins in cereal crops (31 varieties of wheat, oats, and barley) was quantitatively determined and used for the comparative characterization of grains. The quantitative PCR has been adapted for the analysis of the target DNA of Alternaria spp., Bipolaris sorokiniana (B. sorokiniana), Fusarium graminearum (F. graminearum), F. culmorum, and F. sporotrichioides fungi, which are often present in the mycobiota of small grain cereals. The content of the DNA of the aggressive pathogen B. sorokiniana was determined using quantitative PCR for the first time. The DNA of Alternaria fungi was found abundantly in all grain samples, but its content in the oat was significantly higher compared to barley and wheat (5 and 9 times higher, respectively). In barley grain, the content of B. sorokiniana DNA was on average significantly higher than in the grains of oats and wheat. All of the analyzed grain samples contained the DNA of F. graminearum while F. culmorum DNA was found in 70% of the oat samples and in all of the samples of barley and wheat. Mycotoxin deoxynivalenol (DON) produced by these fungi was detected in all the analyzed cereal grains in a range from 77 to 4,133 μg/kg. The DNA of F. sporotrichioides was detected in 70% of oats and 50% of barley samples but was not found in wheat. The T-2 toxin produced by this fungus was detected in 45% of all samples within a range from 2 to 89 μg/kg. The statistically significant positive correlation with the Pearson correlation coefficient (r) equal to 0.49 (p<0.05) was observed between the content of F. graminearum DNA and the amount of DON in the grain samples. Another significant positive correlation (r=0.72, p<0.01) was found between the DNA contents of Alternaria fungi and F. sporotrichioides in the grain samples. This leads to the suggestion that conditions for the growth of these fungi in grain substrates are similar.

Resistance of oat breeding lines to grain contamination with Fusarium langsethiae and T-2/HT-2 toxins

“…To evaluate the fungal infection and species composition of oat grain mycobiota, 100 seeds of each genotype were surface sterilized in 5 % sodium hypochlorite and washed with sterilized water. Then, grains were placed on potato sucrose agar medium (PSA) in Petri dishes (Orina et al, 2018), and incubated in the dark at 24 °С in an MIR-254 thermostat (Sanyo, UK). After seven days, the number and species diversity of fungi isolated from the grain were registered.…”

Microbiological quality of grain cultivated in the North Caucasus region in 2019

“…DNA from flour samples was isolated with a reagent kit (TermoFisher Scientific, Lithuania). TaqMan quantitative PCR (qPCR) was performed using specific primers and probes for the detection of the DNA of Fusarium fungi producing trichothecene mycotoxins (Tri-Fusarium) [22] and the DNA of Alternaria fungi [23]. The reactions were carried out in a 20 μl volume containing 10 μl of 2× TaqMan Master Mix (AlkorBio, Russia), each primer at 300 nM, a fluorescent probe at 100 nM (Evrogen, Russia) and 2 μl of the corresponding DNA solution.…”