In vivo and In vitro evaluation of the antifungal activity of the PGPR Bacillus amyloliquefaciens RaSh1 (MZ945930) against Alternaria alternata with growth promotion influences on Capsicum annuum L. plants

Abstract

Alternaria alternata that threatens pepper production and causes major economic harm is responsible for the leaf spot/blight disease. Chemical fungicides have been widely employed; unfortunately, fungicidal resistance is a current concern. Therefore, finding new environmentally friendly biocontrol agents is a future challenge. One of these friendly solutions is the use of bacterial endophytes that have been identified as a source of bioactive compounds. The current study investigates the in vivo and in vitro fungicidal potential of Bacillus amyloliquefaciens RaSh1 (MZ945930) against pathogenic A. alternata. In vitro, the results revealed that RaSh1 exhibited strong antagonistic activity against A. alternata. In addition to this, we inoculated pepper (Capsicum annuum L.) plants with B. amyloliquefaciens RaSh1 and infected them with A. alternata. As a result of A. alternata infection, which generated the highest leaf spot disease incidence (DI), the plant's growth indices and physio-biochemical characteristics significantly decreased, according to our findings. Our results also showed the abnormal and deformed cell structure using light and electron microscopy of A. alternata-infected leaves compared with other treatments. However, DI was greatly reduced with B. amyloliquefaciens RaSh1 application (40%) compared to pepper plants infected with A. alternata (80%), and this led to the largest increases in all identified physio-biochemical parameters, including the activity of the defense-related enzymes. Moreover, inoculation of pepper plants with B. amyloliquefaciens RaSh1 decreased electrolyte leakage by 19.53% and MDA content by 38.60% as compared to A. alternata infected ones. Our results show that the endophyte B. amyloliquefaciens RaSh1 has excellent potential as a biocontrol agent and positively affects pepper plant growth.

Keywords: Antioxidant defense; Bacillus amyloliquefaciens RaSh1; Electron microscopy; Fungicides; Leaf spot disease; Plant growth.

Fig. 1

Morphological abnormalities in the mycelia of Alternaria sp upon interaction with B. amyloliquefaciens RaSh1 under the light microscope. Images A and B Untreated (control) Alternaria sp spores and mycelia. C Dual culture plate method showing inhibition of Alternaria sp. by B. amyloliquefaciens RaSh1. D Swelling and deformity of Alternaria sp mycelia treated with B. amyloliquefaciens RaSh1. E Untreated (control) Alternaria sp. cultivated on PDA media F Alternaria sp. cultivated on PDA media amended with 0.2% Thiram after 5 days of growth

Fig. 2

Phylogenetic tree of the 18S rRNA genes for A. alternata RaSh3 (OK053809.1) isolate and the others presented on GenBank based on the DNA sequence

Fig. 3

A Disease symptoms on pepper plant leaves infected with A. alternata (T2) and sprayed with Thiram (T3) or inoculated with B. amyloliquefaciens RaSh1 (T5). B Disease incidence percent (DI, %)

Fig. 4

Effect of B. amyloliquefaciens RaSh1 and A. alternata on different pigment fractions; A Chl a, B Chl b and C Carotenoids of pepper plants. *The values are the means of 10 replicates ± SE (n = 10). The same letter above each column indicates no significant difference between the treatments (p ≤ 0.05) as determined by Duncan’s multiple range test. Treatments: T1—control pepper (uninoculated), non-diseased; T2— infected with A. alternata, diseased; T3—infected with A. alternata and sprayed with Thiram (0.2%), diseased; T4—inoculated with B. amyloliquefaciens RaSh1), non-diseased; T5—inoculated with B. amyloliquefaciens RaSh1 and infected with A. alternata

Fig. 5

Effect of B. amyloliquefaciens RaSh1 and A. alternata on water status (%); A relative water content (RWC), B water content (WC) and C water saturation deficit (WSD) of pepper plant leaves under different treatments. *The values are the means of 10 replicates ± SE (n = 10). The same letter above each column indicates no significant difference between the treatments (p ≤ 0.05) as determined by Duncan’s multiple range test. Treatments: T1— control pepper (uninoculated), non-diseased; T2—infected with A. alternata, diseased; T3—infected with A. alternata and sprayed with Thiram (0.2%), diseased; T4—inoculated with B. amyloliquefaciens RaSh1), non-diseased; T5—inoculated with B. amyloliquefaciens RaSh1 and infected with A. alternata

Fig. 6

Effect of B. amyloliquefaciens RaSh1 and A. alternata on A electrolyte leakage (EL) and B membrane stability index (MSI) of pepper plant leaves under different treatments. *The values are the means of 10 replicates ± SE (n = 10). The same letter above each column indicates no significant difference between the treatments (p ≤ 0.05) as determined by Duncan’s multiple range test. Treatments: T1—control pepper (uninoculated), non-diseased; T2— infected with A. alternata, diseased; T3—infected with A. alternata and sprayed with Thiram (0.2%), diseased; T4—inoculated with B. amyloliquefaciens RaSh1), non-diseased; T5—inoculated with B. amyloliquefaciens RaSh1 and infected with A. alternata

Fig. 7

Effect of B. amyloliquefaciens RaSh1 on A lipid peroxidation (nmol/g Fwt) and the antioxidant activity of B CAT and C PPO in A. alternata infected pepper plants. *The values are the means of 10 replicates ± SE (n = 10). The same letter above each column indicates no significant difference between the treatments (p ≤ 0.05) as determined by Duncan’s multiple range test. Treatments: T1—control pepper (uninoculated), non-diseased; T2—infected with A. alternata, diseased; T3—infected with A. alternata and sprayed with Thiram (0.2%), diseased; T4—inoculated with B. amyloliquefaciens RaSh1), non-diseased; T5—inoculated with B. amyloliquefaciens RaSh1 and infected with A. alternata

Fig. 8

Effect of B. amyloliquefaciens RaSh1 and A. alternata on A DPPH radical scavenging activity and B IC50 of methanolic extracts of the different treatments: ASA Ascorbic acid; T1— control pepper (uninoculated), non-diseased; T2—infected with A. alternata, diseased; T3—infected with A. alternata and sprayed with Thiram (0.2%), diseased; T4—inoculated with B. amyloliquefaciens RaSh1), non-diseased; T5—inoculated with B. amyloliquefaciens RaSh1 and infected with A. alternata

Fig. 9

Light microscopy images showing the anatomy of pepper leaves A and B Necrotized inoculation sites of pepper leaves infected with A. alternata C Uninfected control leaves and D Pepper leaves infected with A. alternata treated with B. amyloliquefaciens RaSh1 (MZ945930) as biocontrol agent. E, epidermis; N, necrosis; P, palisade mesophyll; S, spongy mesophyll; V.B, vascular bundle

Fig. 10

Transmission electron micrographs showing normal lenticular-shaped chloroplast with typical thylakoid and grana structures, and small starch grains in uninfected leaf (Control). Ch chloroplast, CW cell wall, G granum, S starch grain, St stroma

Fig. 11

Transmission electron micrographs; A–C showing disorganization of chloroplast structure in the mesophyll cells of pepper leaves infected with A. alternata showing large starch grains and numerous plastoglobules, destroyed outer and inner membranes and collapsed grana and thylakoids. D–F showing chloroplast with preserved thylakoid and grana system and large starch grains in infected pepper leaves and inoculated with B. amyloliquefaciens RaSh1 (MZ945930) as biocontrol agent. Ch chloroplast, CW cell wall, G collapsed granum, P plastoglobule, S starch grain, St stroma