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Abstract

Nanodiagonastic methods in plant pathology are used for enhancing detection and identification of different plant pathogens and toxigenic fungi. Improvement of the specificity and efficiency of the polymerase chain reaction (PCR) by using some nanoparticles is emerging as a new area of research. In the current research, silver, zinc, and gold nanoparticles were used to increase the yield of DNA for two plant pathogenic fungi including soil-borne fungus Rhizoctonia solani and toxigenic fungus Alternaria alternata. Gold nanoparticles combined with zinc and silver nanoparticles enhanced both DNA yield and PCR products compared to DNA extraction methods with ALB buffer, sodium dodecyl sulfate, ALBfree from protinase K, ZnNPs and AgNPs. Also, by using ZnNPs and AgNPs the DNA yield was enhanced and the sensitivity of random amplified polymorphic DNA (RAPD) PCR products was increased. Application of nanomaterials in the PCR reaction could increase or decrease the PCR product according to the type of applied nanometal and the type of DNA template. Additions of AuNPs to PCR mix increased both sensitivity and specificity for PCR products of the tested fungi. Thus, the use of these highly stable, commercially available and inexpensive inorganic nano reagents open new opportunities for improving the specificity and sensitivity of PCR amplicon, which is the most important standard method in molecular plant pathology and mycotoxicology.
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Abstract

Genetically modified Bt cotton (Gossypium hirsutum) leaves with typical symptoms of Alternaria early blight disease resembling that of tomato and potato were observed in the main cotton growing schemes in Sudan. Symptoms on leaves appeared as either brown 2leaf spot with gray centers or leaf blight with concentric rings. Pathogenicity tests using isolates with both symptoms showed that the isolated fungi were highly pathogenic to both G. hirsutum and G. barbadense cotton varieties. Alternaria alternata isolated from infected tomato and potato leaves with early blight symptoms was included for comparison. Microscopic examination showed that the mean length of conidia from cotton, tomato and potato isolates ranged from 26.25 to 45.45 μm, while the width ranged from 9.56 to 13.64 μm. The mean number of transverse septa among all isolates was 3.4 to 5.7 and the peak length ranged from 3.75 to 7.8 μm. Based on morphological characteristics the two isolates from cotton were identified as A. alternata. Genomic DNA was extracted directly from fungal cultures grown on potato dextrose agar (PDA) plates using a Zymo Research Quick DNA kit. A species-specific primer using the internal transcribed spacer ribosomal DNA (ITS rDNA) PCR scoring indicated the presence of A. alternata using primer pair ITS4/ITS5. Amplifications of the internal transcribed spacer region of 600 bp revealed 100% identity of the isolated fungus from cotton with A. alternata from tomato and potato. These data oblige us to reconsider the presence of A. alternata in the four main cotton growing schemes in Sudan while these symptoms have always been described for tomato and potato early blight disease.
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