Tissue-specific synergistic bio-priming of pepper by two Streptomyces species against Phytophthora capsici

Abstract

Among several studied strains, Streptomyces rochei IT20 and S. vinaceusdrappus SS14 showed a high level of inhibitory effect against Phytophthora capsici, the causal agent of pepper blight. The effect of two mentioned superior antagonists, as single or combination treatments, on suppression of stem and fruit blight diseases and reproductive growth promotion was investigated in pepper. To explore the induced plant defense reactions, ROS generation and transcriptional changes of selected genes in leaf and fruit tissues of the plant were evaluated. The plants exposed to the combination of two species responded differently in terms of H2O2 accumulation and expression ratio of GST gene compared to single treatments upon pathogen inoculation. Besides, the increment of shoot length, flowering, and fruit weight were observed in healthy plants compared to control. Likely, these changes depended on the coordinated relationships between PR1, ACCO genes and transcription factors WRKY40 enhanced after pathogen challenge. Our findings indicate that appropriate tissue of the host plant is required for inducing Streptomyces-based priming and relied on the up-regulation of SUS and differential regulation of ethylene-dependent genes.

Fig 1. IT20, SS14, IT20+SS14, and control (PC) in dual culture assay; and inhibition of mycelial growth and germination of Phytophthora capsici after six days of incubation.

Fig 2

Disease scales (A) and disease progress (B) related to chemical (BABA), biological (S. vinaceusdrappus SS14 or S. rochei IT20) and combination of biological treatments (IT20+SS14) from 7 to 28 days after inoculation of Phytophthora capsici (PC).

Fig 3. H₂O₂ production in leaves and fruits of chemical (BABA), biological (S. vinaceusdrappus SS14 or S. rochei IT20) and combination (IT20+SS14) treated plants after seven days of pathogen inoculation respectively.

Same letters represent non-significant difference according to Duncan’s Multiple Range Test (P < 0.05).

Fig 4

The relative level of gene expression (fold) determined by qRT-PCR of eight target genes including Glutathione-S-transferase (A), Sucrose synthase (B), PR1 (C), PR10 (D), WRKY40 (E), WRKY53 (F), ERF (G), and ACC oxidase (H) versus reference control (Actin gene) in leave tissues of pepper treated with chemical (BABA), biological (S. vinaceusdrappus SS14 or S. rochei IT20) and combination (IT20+SS14) after seven days inoculation with PC/ PDA plugs. Untreated and non-inoculated plants considered as control (C). Standard error represents for three biological replicates. Positive values of fold change indicate up-regulation while negative values have been known for the down-regulated genes. The values marked with an asterisk are significantly different from control at P< 0.05.

Fig 5

The relative level of gene expression (fold) determined by qRT-PCR of eight target genes including Glutathione-S-transferase (A), Sucrose synthase (B), PR1 (C), PR10 (D), WRKY40 (E), WRKY 53 (F), ERF (G), and ACC oxidase (H) versus reference control (Actin gene) in fruit tissues of pepper treated with chemical (BABA), biological (S. vinaceusdrappus SS14 or S. rochei IT20) and combination (IT20+SS14) after seven days inoculation with PC/ PDA plugs. Untreated and non-inoculated plants considered as control (C). Standard error represents for three biological replicates. Positive values of fold change indicate up-regulation while negative values have been known for the down-regulated genes. The values marked with an asterisk are significantly different from control at P< 0.05.

Fig 6

Disease severity (A) and disease index (B) of fruits inoculated with the zoospore suspension of Phytophthora capsici (5*10⁶/ml) on pre-inoculated (left) and non-inoculated (right) after five days of inoculation. The same letters represent non-significant difference according to Duncan’s Multiple Range Test (P < 0.05). PC: inoculated control and C: non-inoculated control.

Fig 7. The tissue-specific expression of target genes of C. annuum visualized using log-2 fold change values in a heat map.

The plants were treated with the chemical (BABA), biological (S. vinaceusdrappus SS14 or S. rochei IT20) and combination (IT20+SS14) after inoculation with PC. Untreated and non-inoculated plants considered as control (C).