Chemical and Molecular Detection of New Strains of Xanthomonas Campestris pv. Vesicatoria and Pseudomonas putida Associated with Tomato Crop

Abstract

The lack of research on bacterial diseases and the infrequency of discovering new strains in Iraq prompted the current investigation to examine the occurrence and seriousness of bacterial spot disease on tomatoes in certain areas of Najaf City during the 2022 agricultural season. Additionally, the aim was to isolate and identify the bacterial species Pseudomonas putida. The results from the pathogenicity test revealed that the Xanthomonas Campestris pv. vesicatoria isolate, X. Campestris 2, demonstrated a higher level of disease severity (74%) on tomatoes compared to other isolates, whereas X. Campestris 18 exhibited the lowest pathogenicity (30.76%). The bacterial isolates were confirmed to be X. Campestris pv. vesicatoria species based on diagnostic keys. Microscopic and macroscopic examination confirmed the presence of Gram-negative rod-shaped bacteria with circular, yellow colonies on NA medium, while on YDC medium, the bacterial growth appeared as shiny, yellow, and moist colonies. The results of biochemical tests indicated that X. Campestris pv. vesicatoria tested positive for certain tests including the Catalase test, Gelatin Hydrolysis, Motility test, and Aesculin Hydrolysis. However, it tested negative for the Oxidase test, Arginine Hydrolysis, KOH Solubility, and Starch hydrolysis. As for P. putida, microscopic examination revealed Gram-negative rod-shaped bacteria. On King’s B agar, the macroscopic examination showed creamy-colored growth, and the bacteria displayed a bright blue color. The test results indicated positive outcomes for the Catalase test, Oxidase test, Arginine Hydrolysis, Motility test, and fluorescence under UV light, appearing as a bright blue color. The bacteria tested negative for Starch hydrolysis, Indole test, Gelatin liquefaction, and KOH Solubility. Molecular diagnosis based on the 16S rRNA gene confirmed the presence of new strains in the analyzed isolates, which were deposited in NCBI with Accession Numbers: OM966903 for X. Campestris bacteria and OM988185 and OM988186 for P. putida isolates. Furthermore, treatment with P. putida 1 exhibited the highest rate of inhibition against the growth of X. Campestris 2, with a diameter of 26.67 mm, followed by P. putida 2 with an inhibition rate of 16.33 mm, compared to the control treatment, which showed no inhibition (0.00 mm).