3.1. Pathogenicity test on potato slices
Five isolates were obtained from naturally diseased potato tubers and were used to identify their pathogenic ability on healthy potato slices. The results of repeated pathogenicity tests showed that all of these isolates were pathogenic and able to produce typical soft rot symptoms with a range of severity from strong to weak. Isolates No 2 and 5 showed highest diseases followed by isolate no 3. The lowest disease was recorded by isolate no 1. The results of the pathogenicity test on the slices are presented in Table 1. From this results isolates no 2 was selected to complete the following experiments.
Table 1: Disease severity index of selected bacterial soft rot isolates.
Isolates No
|
Disease severity %
|
1
|
25 c
|
2
|
45 a
|
3
|
20 c
|
4
|
22 c
|
5
|
40 b
|
Values in the column followed by different letters indicate significant differences among treatments according to a least significant differences test (P = 0.05).
3.2. Identification of pathogens
According to the in vitro screening, the most virulence isolate Pcc 2 was molecularly identified using 16S rRNA gene partial sequencing. The obtained sequence was submitted to GenBank under accession number PP422427. A phylogenetic analysis was performed using the maximum likelihood method in BLAST pairwise alignments. The isolate was identified as Pectobacterium carotovorum with 100% identity with MT510006 (Fig. 1).
3.1. Characterization of AgNPs
The FTIR spectrum used to determine the vibrational frequency of stretching and bending modes of the molecules of MgO nanoparticles which showed in Fig. 2. The results of FTIR analysis of this study show different stretches of bonds shown at different peaks; 3405.02—N–H stretch, 2927.92—single aldehyde, 2857.20—C–H; O–H,, 1656.34—C = C, and 1127.23—C = O.. The weaker band at 1598.68 cm− 1 corresponds to amide I arising due to carbonyl stretch in proteins. The peak at 1049.61 cm− 1 corresponds to C–N stretching vibration of the amine. the peak near 575.25cm− 1 assigned to CH out of plane bending vibrations are substituted ethylene systems –CH = CH (cis).The result of TEM images indicated that the biosynthesized AgNPs have a spherical shape with particle size 25.6 nm (Fig. 3).
3.2. Antibacterial activity
First different concentrations of AgNPs were tested for its antibacterial activity on NA plates then, the inhibition zone (cm) was measured which was 0.8 cm, 1.6 cm 2.5 cm for concentration 50,100 and 200 respectively (Fig. 4). For MIC assay, 200 µg/ml was found to kill all bacteria after 48 h incubation which introduced at tubes and 94 wells microplate (Fig. 4) which exhibited inhibition rate at OD 600 (77.8%) after 48 h. AgNPs had high antibiofilm of 0.87, 0.43 and 0.28 nm reductions at 50, 100 and 200 µg/ml, if compared with control (1.22 nm) after 48 h. Motility assay performed on 0.3% agar NA showed a swimming inhibition was 13.00, 8.00 and 5.00 mm % for 50, 100 and 200 µg/ml respectively when compared to the control 17.33 mm (Fig. 5).
3.4. In vivo antibacterial tuber slice assay
Compared to control tubers infected with P. carotovorum, potato tubers treated with both nanoparticles and P. carotovorum recovered substantially more. (Fig. 6). In brief, the suppression rate of P.carotovorum was (12.67, 45 and 98.67%) at different concentration (50,100 and 200µg/ml) respectively.