Bacillus licheniformis strain POT1 mediated polyphenol biosynthetic pathways genes activation and systemic resistance in potato plants against Alfalfa mosaic virus

Abstract

Alfalfa mosaic virus (AMV) is a worldwide distributed virus that has a very wide host range and causes significant crop losses of many economically important crops, including potato (Solanum tuberosum L.). In this study, the antiviral activity of Bacillus licheniformis strain POT1 against AMV on potato plants was evaluated. The dual foliar application of culture filtrate (CF), 24 h before and after AMV-inoculation, was the most effective treatment that showed 86.79% reduction of the viral accumulation level and improvement of different growth parameters. Moreover, HPLC analysis showed that a 20 polyphenolic compound was accumulated with a total amount of 7,218.86 and 1606.49 mg/kg in POT1-treated and non-treated plants, respectively. Additionally, the transcriptional analysis of thirteen genes controlling the phenylpropanoid, chlorogenic acid and flavonoid biosynthetic pathways revealed that most of the studied genes were induced after POT1 treatments. The stronger expression level of F3H, the key enzyme in flavonoid biosynthesis in plants, (588.133-fold) and AN2, anthocyanin 2 transcription factor, (97.005-fold) suggested that the accumulation flavonoid, especially anthocyanin, might play significant roles in plant defense against viral infection. Gas chromatography-mass spectrometry (GC-MS) analysis showed that pyrrolo[1,2-a]pyrazine-1,4-dione is the major compound in CF ethyl acetate extract, that is suggesting it acts as elicitor molecules for induction of systemic acquired resistance in potato plants. To our knowledge, this is the first study of biological control of AMV mediated by PGPR in potato plants.

Figure 1

A photograph showing the disease symptoms on potato leaves infected with AMV at 50 days post inoculation. Where A: Mock-treated plants (T1), B: plants inoculated with AMV only (T2), C: plants treated with CF 24 h before inoculation of AMV (T3) and D: plant treated with CF, 24 h before inoculation of AMV and 24 h after inoculation with AMV (T4).

Figure 2

A histogram showing the relative expression level of AMV-CP gene in AMV-infected potato plants at 21 days post inoculation. Where, T1 = Mock-treated plants (Control), T2 = plants inoculated with AMV only, T3 = plants treated with CF 24 h before inoculation of AMV, and T4 = plant treated with CF, 24 h before inoculation of AMV and 24 h after inoculation with AMV. Columns represent mean value from three biological replicates and bars indicate Standard Deviation (± SD). Significant differences between samples were determined by one-way ANOVA using CoStat software. Means were separated by Least Significant Difference (LSD) test at P ≤ 0.05 levels and indicated by small letters. Columns with the same letter means do not differ significantly.

Figure 3

A histogram showing the relative transcriptional expression levels of polyphenol (phenylpropanoid, chlorogenic acid, and flavonoid) biosynthetic pathways genes in AMV-inoculated potato plants at 21 days post inoculation. Where, T1 = Mock-treated plants (Control), T2 = plants inoculated with AMV only, T3 = plants treated with CF 24 h before inoculation of AMV, and T4 = plant treated with CF, 24 h before inoculation of AMV and 24 h after inoculation with AMV. Columns represent mean value from three biological replicates and bars indicate Standard Deviation (± SD). Significant differences between samples were determined by one-way ANOVA using CoStat software. Means were separated by Least Significant Difference (LSD) test at P ≤ 0.05 levels and indicated by small letters. Columns with the same letter means do not differ significantly.

Figure 4

HPLC chromatograms of polyphenolic compounds identified in ethanol extract of potato leaves at 21 days post inoculation of different treatments. Where, T1 = Mock-treated plants (Control), T2 = plants inoculated with AMV only, T3 = plants treated with CF 24 h before inoculation of AMV, and T4 = plant treated with CF, 24 h before inoculation of AMV and 24 h after inoculation with AMV.

Figure 5

A histogram showing comparison of log of relative accumulation levels of polyphenolic compounds identified in ethanol extract of potato leaves at 21 days post inoculation of different treatments. Where, T1 = Mock-treated plants (Control), T2 = plants inoculated with AMV only, T3 = plants treated with CF 24 h before inoculation of AMV, and T4 = plant treated with CF, 24 h before inoculation of AMV and 24 h after inoculation with AMV.

Figure 6

Histogram showing Gas chromatography-mass spectrometry (GC-MS) fractionation of ethyl acetate extract of Bacillus licheniformis strain POT1.