Identification and green synthesis of potato dry rot Fusarium falciforme silver nanoparticles, characterization and antibacterial activity

Abstract

Potato tubers collected from different areas showed the prevalence of dry rot with characteristic white mycelia like symptoms of Fusarium pathogen. Fusarium species from diseased tubers were isolated based on morphological features. 18 S-Internal Transcribed Spacer (ITS) and Translation Elongation factor 1-α (TEF1-α) based analysis followed by phylogenetic tree constructed using ITS, TEF1-α and RNA polymerase II subunit B (RPB2) identified Fusarium isolates as Fusarium verticilloides, Fusarium soloni, Fusarium falciforme and Fusarium oxysporum. According to our knowledge, this study is the first report of the occurrence of Fusarium falciforme causing dry rot on potato tubers in Pakistan. The pathogenicity test confirmed the re-appearance of dry rot symptoms with Fusarium falciforme on potato tuber. The formation of Fusarium falciforme Ag NPs was confirmed using UV-Visible spectroscopy, X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and Field-emission scanning electron microscopy (FE-SEM) analysis. The synthesized Ag NPs showed color change with Fusarium falciforme and accordingly, UV peak was observed at 431 nm. The XRD revealed Ag based specific angles at 36.14°, 44.26°, 64.42° and 77.44° corresponding to face cubic structure. Similarly, FTIR band absorption at 3297 cm^-1, 1626 cm^-1 and 1057 cm^-1 ascribed the presence of various biomolecules with O-H, N-H and C-N stretching vibrations based on Ag NPs synthesis. SEM indicated 29 nm synthesized mainly spherical Ag NPs and EDS analysis showed the presence of Ag in NPs. Maximum zone of inhibition with Fusarium falciforme Ag NPs was observed against Pseudomonas aeruginosa, Escherichia coli, Pseudomonas syringae and Staphylococcus aureus at 0.05 LSD significance.

Keywords: Fusarium falciforme; Antibacterial; Potato; Silver nanoparticles.

Fig. 1

Dry rot diseased tubers from KP Pakistan; (A) Batakundi and (B) Mansehra (C) Haripur and (D) Abbottabad showing clear symptoms of dry rot of potato

Fig. 2

Fusarium species with (A) TEF1-α and (B) ITS PCR applied to dry rot samples isolated from diseased potato tubers

Fig. 3

Fusarium cultures isolated from Batakundi, Manshera and Haripur, KP, Pakistan (A–D) ZHW3 Fusarium verticilliodies (NCBI accession number ITS PV211456, TEF1-α PQ775192, RPB2 PV606467); (E–H) ZHW4 Fusarium solani (NCBI accession numbers ITS PV211459, TEF1‐α PQ775194, RPB2 PV626798); (I–L) ZHW5; Fusarium falciforme (NCBI under accession numbers ITS PV211464, TEF1‐α PQ775196, RPB2 PV626797); (M–P) ZHW6 Fusarium oxysporum (NCBI accession numbers ITS PV211468, TEF1‐α PQ775198, RPB2 PV606468)

Fig. 4

Maximum likelihood phylogenetic tree based on concatenated sequences using ITS, TEF1-α and RPB2 show the phylogenetic relationships of Fusarium isolates ZHW3, ZHW4, ZHW5 and ZHW6 with selected reference strains. The tree was inferred using IQ-TREE v2.2.0 under the HKY + I + F model, which was selected by Partition Finder as the best-fit model. Support values from the Shimodaira-Hasegawa-like approximate likelihood-ratio test (SH-aLRT) are shown at the nodes (values ≥ 80% are displayed). Neonectria ditissima (CBS100316) was used as the outgroup. Isolates ZHW5 (marked with a red star) and F. falciforme (NCS54) are highlighted in red. The scale bar indicates 0.1 nucleotide substitutions per site. Clade 1, Clade 2, and Clade 3 represent major phylogenetic groupings

Fig. 5

Pathogenicity test to confirm Fusarium symptoms on potato tubers

Fig. 6

Synthesis of Ag NPs. (A) Fungal Biomass (B) Fungal Filtrate (C) Fusarium Ag NPs

Fig. 7

UV-Visible spectrum of Fusarium Ag NPs

Fig. 8

XRD pattern of Ag NPs

Fig. 9

FTIR pattern of synthesized Ag NPs

Fig. 10

SEM images of (A) Ag NPs and (B) EDS of Ag NPs

Fig. 11

Fusarium falciforme Ag NPs antibacterial activity at 30, 60 and 90 ug/uL concentration (A) Pseudomonas aeruginosa (B) Escherichia coli (C) Pseudomonas syringae (D) Staphylococcus aureus. Ampicillin is used as positive control and Fusarium filtrate (Ff) at 60 ug/uL

Fig. 12

Antibacterial activity of Ag NPs. Zone of inhibition with Ag NPs at 30, 60 and 90 ug/uL Ag NPs (A) Pseudomonas aeruginosa (B) Escherichia coli (C) Pseudomonas syringae DC3000 (D) Staphylococcus aureus. Data is represented as the means ± SD from 3 replicates. Different letters represent significant differences at p ≤ 0.05 Fisher LSD one way ANOVA