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Abstract
The aim of this work was to isolate root-nodule bacteria from different leguminous plants (one wild and 5 cultivated herb legumes), that grow in cultivated lands in Beni-Suef governorate, Egypt, and to determine the phenotypic (e.g. utilization of carbon and nitrogen sources, tolerance to salt, heat, heavy metals, and acid, and antibiotic resistance), and molecular (REP-PCR patterns and amplification of 16S rDNA) characteristics of these bacteria. The characteristics of these rhizobia, as well as six reference strains of the genera Rhizobium and Mesorhizobium, were investigated in order to establish a polyphasic taxonomy of these rhizobia. Also, 16S rDNA-based phylogenetic trees and pair wise alignments of these isolates with the type strains (T strains) in the gene bank were obtained to get precise homologies of these isolates to the nearest T strains.
The results obtained in this study could be summarized in the following points:
* Rhizobia (68 isolates) were isolated from 6 leguminous plants namely: Cicer arietinum (11 isolates), Vicia faba (11 isolates), Pisum sativum (12 isolates), Phaseolus vulgaris (12 isolates), Lens esculentus (11 isolates), and Trifolium resupinatum (11 isolates). After their isolation, isolates were confirmed as rhizobia by re-nodulating them to their host legumes. All isolates succeeded to renodulate their corresponding hosts except two isolates; BSTR8 from T. resupinatum, and BSLE from L. esculentus.
* Rhizobial isolates displayed various colony characteristics. Some colonies were circular, elevated, shiny, and milky or opaque when grown on YMA medium. The formed colonies had diameter ranged between 2-5 mm after 3-5 days of incubation at 28 °C.
* Isolates were phenotypically characterized and the following results were obtained:
1) All isolates from various hosts were able to utilize glucose and mannitol as carbon sources. About 78% utilized sucrose and lactose while inositol was the least carbon source (only 38% of isolates utilized it) utilized.
2) About 80% of isolates in this study utilized serine as a sole source of nitrogen, 70% utilized asparagin, and 68% utilized glycine.
3) All isolates succeeded to grow at 1% and 2% NaCl. Represenatative isolates from each host plant, except from V. faba, survived at 3% NaCl, and about 15% survived at 4% NaCl. Only one isolate (BSTR8) from T. resupinatum survived at 5% NaCl.
4) All isolates succeeded to grow at 6-8 pH values. At pH 9, about 70% of isolates grew. This included the whole isolates form both C. arietinum and P. vulgaris and about 80% of the isolates from P. sativum and T. resupinatum. At pH 10, eight (out of 11) isolates from C. arietinum, ten (out of 12) from P. vulgaris, and two (out of 11) from L. esculentus survived.
5) The optimum temperature for the growth of all isolates was in the range 20-35ºC. Below and beyond this range, the isolates showed variations in their growth. All isolates were able to grow at a 20-35ºC temperature range. 50% of isolates, including all isolates from C. arietinum, and seven isolates (out of 11) from V. faba survived at 40ºC. Only 20% of isolates survived at 45ºC.
6) All isolates showed positive catalase enzyme test, 82% showed positive gelatinase enzyme test and 70% showed positive urease enzyme test.