ABSTRACT
Powdery mildew is an omnipresent disease that reduces the yield and quality of pea crops (Pisum sativum L.). To examine the powdery mildew pathogen's morphological, molecular, and genetic diversity, we collected samples of powdery mildew-affected pea crops from ten distinct locations in the Nilgiris district of Tamil Nadu, India. The pathogen Erysiphe pisi was identified morphologically based on anamorphic characters. Molecular identification of E. pisi isolates was befitted by targeting the internal transcribed spacer (ITS) region of rDNA and specific primers of powdery mildew fungi. The genetic variation between ten different E. pisi isolates collected from topographically distinct mountainous areas was studied using random amplified polymorphic (RAPD). Based on its morphological characteristics, the powdery mildew fungus presented high similarities to E. pisi. Molecular characterization of the ITS rDNA of E. pisi produced 650 bp nucleotides, PMITS (powdery mildew-internal transcribed region) primers produced 700 bp nucleotides, and an Erysiphe specific ITS primer pair amplified and synthesized 560 bp nucleotides. According to the findings, the collected E. pisi strains exhibited a low level of genetic diversity and only a slight differential in virulence on the host. In the study, E. pisi isolates from Anumapuram, Emerald Valley, Indira Nagar, and Thuneri showed a greater disease incidence in the natural field conditions and shared the same genetic lineage with other isolates in UPGMA hierarchical cluster analysis based on RAPD markers. There was no evidence of a link between the occurrence of the disease and these grouped populations.
ABSTRACT
BACKGROUND: The Clear Safety Salmonella method was modified to improve sample preparation, PCR reagents, library preparation, flow cell quality control, library loading mix, priming mix, and sequencing kit reagents and steps. OBJECTIVE: To evaluate the modified Clear Safety Salmonella method (manual and automated) via independent and method developer validation studies according to current AOAC INTERNATIONAL Validation Guidelines. METHOD: Performance of the modified Clear Safety Salmonella method (manual and automated) was assessed for selectivity (using 105 inclusive and 30 exclusive strains), probability of detection in matrixes, product consistency, stability, and robustness. The modified Clear Safety Salmonella method was compared with the appropriate reference method for Salmonella detection on 4 inch × 4 inch stainless steel environmental surfaces, and in chicken carcass rinse (30 mL), raw ground chicken (375 g), dry pet food (375 g), and ready-to-eat deli turkey breast (375 g). RESULTS: The modified Clear Safety Salmonella method (manual and automated) demonstrated no statistically significant differences between the candidate and reference method probability of detection or between the presumptive and confirmed results for all target food matrixes and the stainless steel surface. Additionally, the modified method (manual and automated) detected all 105 inclusivity organisms and excluded all 30 exclusivity organisms. The product consistency and kit stability studies showed no statistical differences between lots or over the term of the kit's shelf life. In robustness studies, changes in enrichment time, diluted sample volume, and sample volume for PCR did not show any statistical difference in terms of assay performance. CONCLUSIONS: The modified Clear Safety Salmonella method (both manual and automated) is statistically equivalent to or better than the reference methods. HIGHLIGHTS: The Clear Safety Salmonella method utilizes PCR amplification and targeted next-generation sequencing technology to selectively detect Salmonella enterica.
