Background and Objective
Legumes, particularly beans, are crucial to food security and soil health. The crops account for approximately 50% of global legume production and are fundamental in meeting the food needs of various societies. Rhizobium bacteria, known for their nitrogen-fixing abilities, serve as valuable symbionts to leguminous plants. As the primary source of biofertilizers, Rhizobium plays a vital role in nitrogen fixation, enhances soil fertility, and helps reduce dependence on chemical fertilizers. The Rhizobium radiobacter aids plants in resisting environmental stresses. This study aims to identify and investigate the phenotypic and genetic diversity of Rhizobium isolates, addressing the limited research on the diversity of these bacteria in the soils of the Middle Zagros region.
Materials and Methods
The bean roots were sampled during the summer flowering stage from the cities of Aleshatar, Azna, Aligudarz, Beyranshahr, and Khorramabad. The samples were transferred to the plant pathology laboratory, where the surfaces of the roots and nodes were washed, and a bacterial suspension was prepared. Subsequently, bacterial isolation was performed on the YEMA culture medium. Phenotypic, biochemical, and nutritional tests were employed to identify the bacteria accurately. Additionally, to enhance the identification accuracy, the 16S rDNA region was amplified and sequenced. Ribosomal DNA restriction enzymes were employed to study diversity at the nucleotide level. This study investigates genetic diversity and identifies superior isolates of this species concerning nitrogen supply, host enhancement, and grain yield increase in beans. Sampling was conducted from the roots of bean plants in the Middle Zagros regions. After the isolation and purification of the isolates, phenotypic-biochemical tests were performed for initial screening. Genetic diversity was determined using polymerase chain reaction (PCR), and the results were analyzed using Ntsys-pc version 2.02 software. The symbiotic efficiency of the identified isolates was investigated under greenhouse conditions using the Akhtar and Sayyad bean cultivars. A total of 14 isolates of R. radiobacter were identified and analyzed for their effectiveness in promoting plant growth and health.
Results
A total of 14 bacterial isolates of R. radiobacter were identified. The bacterial isolates of R. radiobacter were identified as gram-negative, oxidase-positive, and catalase-positive. These obligate aerobic organisms could grow on culture media containing 3% salt. The isolates demonstrated positive results in Tween 80 hydrolysis, esculin hydrolysis, alkalinization of Litmus Milk, levan production, and ketolactose production. However, they were negative for starch hydrolysis, gelatin hydrolysis, nitrate reduction, indole production, and fluorescent dye tests. Isolate Rb194 exhibited a 100% identity to the R. radiobacter species. The analysis of ARDRA using HpaII and RsaI enzymes demonstrated the genetic diversity among the isolates. During a greenhouse experiment aimed at investigating the efficiency of symbiosis with two bean cultivars, the results were as follows: for the Akhtar cultivar, isolates Rb180, Rb184, and Rb187 exhibited the highest efficiency of symbiosis, with the highest shoot dry weight attributed to isolate Rb187.
Additionally, isolates Rb182, Rb189, and Rb190 could not produce nodules. For the Sayyad cultivar, isolates Rb180 and Rb182 produced the highest shoot dry weight; notably, the symbiotic efficiency of Rb180 exceeded 100%, while isolates Rb189 and Rb190 could not produce nodules. The PCR and restriction enzyme analysis results revealed distinct bands ranging from 150 to 2000 base pairs.
Discussion
The rhizobium isolates exhibited significant phenotypic and genetic diversity. The observed differences may be attributed to various environmental factors. The differing efficiency of the isolates in symbiosis with bean plants underscores the importance of the genetic diversity of these bacteria. Genetic diversity analyses revealed substantial differences. Furthermore, the results of the variance analysis regarding the symbiotic efficiency of the isolates demonstrated significant differences in the number and weight of nodules, shoot weight, and nitrogen fixation rates. Differential phenotypic tests are proposed as a rapid and effective method for identifying R. radiobacter. The ARDRA technique is highlighted as a suitable tool for investigating the genetic diversity of these species. Genetic diversity directly correlates with nitrogen fixation ability, climatic conditions, and bacterial activity.