Nanotools for molecular identification two novels Cladosporium cladosporioides species (Cladosporiaceae) collected from tomato phyloplane

Abstract

Magnetic nanoparticles (MNPs) consisting of magnetite (Fe 3 O 4 ) are promising as nanodiagnostic tools in plant pathology. Magnetic nanoparticles (MNPs) were produced using hydrothermal protocols. MNPs size diameter and size distribution were characterized by transmission electron microscopy (TEM) and dynamic light scattering (DLS). Fe 3 O 4 nanoparticles are monodispersible and spherical with an average diameter of 82 nm. Dynamic light scattering analysis of the same samples revealed that the synthesized MNPs were highly monodispersed and had a hydrodynamic diameter ranging from 100 to 201. Fungal DNA was extracted using MNPs in comparison with the conventional sodium dodecyl sulfate (SDS) method in the context of quality, quantity and timing process. The quality and yields of the isolated DNA from all Cladosporium strains using magnetic nanoparticles were higher compared to the DNA isolation method via sodium dodecyl sulfate (SDS). PCR using specific primers targeting ITS and Actin genes were amplified 100% of varying sized gene fragments, verifying the high quality of the isolated DNA. Forty seven fungal isolates belonging to the Cladosporium cladosporioides complex were observed and phylogenetically evaluated on the basis of DNA sequences of the internal transcribed spacer regions ITS1 and ITS2, in addition to partial actin and translation elongation factor 1-α gene sequences. Maximum likelihood phylogenetic analyses were performed for the combined data set (ITS + ACT + TEF) using RAxML. The three Cladosporium strains were isolated from tomato in Saudi and Egypt was identified very similar to Cladosporium asperulatum and Cladosporium myrtacearum based on their molecular phylogenetic characteristics. DNA isolation using magnetic nanoparticles will expectedly be used commonly both in plant pathology laboratories and in the nanobiotechnology industry.