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The research was carried out in the plant Bacterial Diseases Laboratory, Faculty of Agriculture, Damascus University, and the Biodiversity Laboratory, Molecular Biology Laboratory, and the Green House in the National Commission for Biotechnology during 2018-2024. The research aimed to isolate rhizobial bacteria from roots of some wild and cultivated legume species. These isolates were identified by biochemical and molecular tests and artificial infection. The genetic relation among the isolates was studied using rep-PCR. and their antagonistic ability have been tested against a local isolate that causes vascular wilt in chickpeas; Fusarium oxysporum f sp. ciceris in vitro and under greenhouse conditions. The research also aimed to optimize production conditions of a local biofertilizer based on selected rhizobia. Leguminous plants (faba bean, chickpea, pea, dandelion, alfalfa, clover, soybean, fenugreek, vetch, and lentil) were collected from seven Syrian governorates; Damascus countryside, Homs, Daraa, Al-Suwayda, Latakia, Hama, and Raqqa. Bacteria were isolated from the root nodules of these plants on yeast agar and Yeast mannitol agar (YMA). Of 109 bacterial isolates, 5 isolates colored red on YMA with Congo red and other 43 that were Gram-positive were excluded. Biochemical and molecular tests and artificial infection for the other 61 isolates showed that 42 of them belong to the Rhizobia group. A cross-infection test was conducted to ensure the specialization of isolates on symbiot plant hosts, Root nodules appeared only on the plant from which the isolate was isolated, which confirmed the specification of bacterial species to their hosts. Accordingly, the isolates were divided into genera; Mesorhizobium symbioses with chickpeas, Rhizobium with faba beans and peas, Bradyrhizobium with soybeans, Ensifer with alfalfa and dandelion. Rep-PCR tests were conducted for only 25 rhizobia isolates. RK30 and RJ23 isolates respectively obtained from root nodules of chickpea plants in Kafr al-Lahf (Suwayda) and Jbab (Daraa) respectively, were used as the reference ones. REP-PCR revealed polymorphism among the bacterial isolates. The similarity between the two reference isolates reached 80%, and among the studied isolates ranged between 37% and 100%, while it ranged from 38% to 83% between these isolates and the reference ones, and the isolate R102 from chickpea plant in Reemet-al-lehef (Suwayda), was the closest to the reference isolate RK30 with similarity of 83%, while the two isolates R45 and R62, from chickpea plants in Wulgha and Kafr al-Lahf-AlSuwayda, were the closest to the reference isolate RJ23 by 79% of similarity, and the correlation between strains isolated from different environments ranged from 55 to 80%. According to Rep-PCR the bacterial isolates of rhizobia symbiotic with legume roots were divided into four groups; The first group included the reference isolates and 9 genotypes of the genus Mesorhizobium from Suwayda, and the second included 4 genotypes of the genus Sinorhizobium/Ensifer that symbiotic with fenugreek, dandelion, and alfalfa plants from Damascus Countryside (Al-Nabk) and Homs (Homs Research Center). The third one had 4 genotypes of the genus Rhizobium, symbiotic with pea, fababean and clover plants from Damascus countryside (Abu Jarash), Homs (Homs Research Center) and Suwayda (Salkhad), the fourth group included two genotypes of the genus Bradyrhizobium, symbiotic with soybean plants, from al-Sahel(Raqqa). The first and fourth groups clusteed accordig to the geographical region, unlike the second and third ones. 61bacterial isolates; 42 isolates of rhizobium (Mesorhizobium, Rhizobium, Bradyrhizobium, and Ensifer), were used to test the antagonistic ability in vitro towards two fungal isolates of Fusarium wilt of chickpea identified locally as (F51 and F63) and obtained from Al-Baath University. 31 isolates showed their ability to inhibit fungal growth; 13 isolates from rhizobia group and 18 from unidentified rhizobacteria. The two isolates; 134 (Rhizobium) and 133 (unidentified rhizobial bacteria) outperformed the other isolates in inhibiting the F63 fungus by 68.89% and 65.93%, respectively, while isolate 61 (Mesorhizobium) inhibited significantly F51 fungus by 53.70%. The three isolates (134 and 133 on bean plants, 61 on chickpea plants) were tested in the greenhouse, and the comparison of all the treatments; (F63×133, F63×134, F51×133, F51×134) with the control of fungus alone (F63 control, F51 control) figured out that the bacteria increased the plant's tolerance to the fungal disease since it led to a significant difference in the severity of the disease and the infection rate, in contrast to the two treatments (F63×61 and F51×61) on the chickpea plant, which achieved a significant difference in increasing some growth indicators and reducing the incidence and severity of the disease. All that revealed the importance of inoculating legume seedlings with appropriate isolates of root nodule bacteria, not only to improve growth indicators, but also to reduce the damage caused by Fusarium vascular wilt disease. Based on the results of identification and in vitro and pot trials antagonism, R134 was selected as the most efficient bacterial isolate, to be inoculated on three types of carrier materials: sieved soil, tourb, perlite, and the bacterial concentration were monitored for 5 months during which they were stored at temperatures different; 4 and 25°C. The results showed that 4°C was superior temperature in preserving the vitality of bacteria for one month compared to the temperature 25°C. A for the concentration of bacteria increased slowly and did not decrease under the recommended concentration 107 until the end of the fourth month, unlike the degree 25°C, at which the concentration increased rapidly then fell below the recommended level in the fourths month period. On the other hand, the sieved soil was the best bacteria-carrier material, followed by perlite and then tourbe. The bacterial concentration reached its highest value during the second month, regardless of the different carrier materials and preservation temperature in the experiment. The results of this study showed the possibility of producing a local biofertilizer by loading bacteria on sieved soil, tourb, or perlite, but it must be tested in field conditions to study its stability in different climatic conditions.
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