Isolation and Diagnosis of the Root Nodule Bacteria Associated with Mung Bean Plant in Gypsiferous Soil and Testing the Promotional Criterion of Isolates

Abstract

The root nodule bacteria are utilized in the production of natural biological fertilizers to achieve clean agriculture by reducing chemical fertilizers. In this study, 35 samples of the root nodules associated with mung bean plants were collected from various agricultural areas of Iraq, Salah Al-Din Governorate on 4\\7\\2022, ten samples isolated by growing them on yeast extract mannitol agar (YEMA) and the phenotypically pure isolates were diagnosed based on the culture, microscopic and biochemical characteristics., The phenotypic diagnosis results showed that The color of the colonies was between white, creamy and yellow, Spherical, convex and smooth, gram-negative, was able to move and pink and light pink on Congo red stain medium and 9 isolates it was fast growing as it gave yellow color on YEMA-BTB bromothymol blue medium except for one isolate it was slow growing as it gave blue color and all isolates unable to grow on Hofer alkaline medium except for one isolate, The efficient isolate was selected in the production of indole acetic acid, chelating compounds and phosphate solubilization,. The results showed that isolates (M3, M4, M5, M6, M7) have a high ability for iron chelation. It was found that the (M6) isolate gave the highest phosphate solubilization and indole production, which showed a phosphorus solubilization of 39.504 mg P.L-1 and an indole production rate of 21.5 μg.ml-1. This was followed by the isolate (M3), which showed a phosphorus solubilization of 23.723 mg P.L-1 and an indole production rate of 17.2 μ.g.ml-1. Molecular diagnosis was performed for five competent isolates in the production of Indole acetic acid, production of chelating compounds, and phosphate solubilization. The isolates were molecularly diagnosed by PCR, the 16SrRNA gene was amplified, then the sequence of nitrogenous bases was analyzed and when matching with the global strains included in the NCBI Genetic Bank website, The results of molecular diagnosis showed that two isolates belong to Rhizobium bacteria. the results showed that (M3) isolate is %99.50 similar to the Rhizobium leguminosarum isolate OTU21_I. and the results showed that (M6) isolate is 99.48% similar to the Bradyrhizobium japonicum strain A3 and 99.96% to the Bradyrhizobium japonicum, strain:NK5 and therefore the isolates is genetically close to Rhizobium leguminosarum strain AE15 and Bradyrhizobium japonicum strain AE14 bacteria and has been recorded in the Global genome bank under accession number OP975690 and version number OP975690.1 for Rhizobium leguminosarum and accession number OP975688 and version number OP975688.1 for Bradyrhizobium japonicum and this record is the first for this bacteria which associated with mung bean in gypsiferous soils in Iraq.