Diversity, Symbiotic and Plant Growth Promoting Properties of Rhizobia and Rhizobacteria of Grass Pea (Lathyrus Sativus L.) From Central Ethiopia: Implication to the Selection and Use of Microbial Inoculants in Low Input Agriculture in Ethiopia

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Addis Ababa University


Grass pea (Lathyrus sativus L.) is widely cultivated for food and feed in some developing countries including Ethiopia. However, its alkaloid content did not attract attention to the research community for its over-exaggerated neuro-lathyrism causing paralysis of limbs making it as one of the most neglected orphan crops in the world. However, the crop is considered as an insurance crop by resource-poor farmers for it produces reliable yields when all other crops fail. The increase in production in several areas in Ethiopia presupposes that it is an important source of protein to the diet of the population and makes it the fifth most important pulse crops in Ethiopia after faba bean, chickpea, field pea and haricot bean in terms of production. This indicates the necessity of looking beyond the prejudices held on the crop for long to expand the frontier of food security and soil fertility against the changing climate in the country. To this end, a study was made to characterize and select rhizobial and rhizobacteria from grass pea from central Ethiopia using standard methods. Isolates were tested for their phenotypic, symbiotic, genetic, in vitro stress tolerance, variations in substrate utilization and PGP properties within the context of their taxonomic characteristics and selection of tolerant isolates for field applications. Based on their preliminary symbiotic effectiveness, stress tolerance (pH tolerance, intrinsic antibiotic resistance (IAR)), PGP (phosphate solubilization, indole acetic acid production, etc), and antagonistic properties (suppression of fungal pathogen, etc), four rhizobial and two rhizobacteria isolates were selected and tested for single and co-inoculation trials of the crop on soil culture under greenhouse conditions. The data showed that all but one rhizobium isolates (49 isolates) were authenticated as root nodule bacteria (renodulating the host variety upon reinoculation) and 86% of the isolates accumulated more than 50% of the shoot dry weight (SDW) (0.273-1.148g/p in relation to N-fetilized control plants indicator of symbiotic effectiveness (SE%) in nitrogen fixation. The inoculated plants also showed variations in nodule number (NN) ranging from 17.7-116/plant, Nodule dry weight (NDW) 0.011g-0.098/plant indicating a seven-fold and ten-fold difference among the isolates. Among the highly symbiotic effective isolates (SDW of >80%), isolates AAUGR- 2, 5, 6, 9, 11, 14, 15, 19, 20, 24, 30 and 50 acquired a wide range of substrate utilization and ecological tolerance under in vitro-conditions and the xv majority of these isolates showed multiple plant growth promoting properties (MPGP) (4-8) ranging from good potential of phosphate solubilization and suppression of fungal pathogens. The PCR amplification of 16SRNA and nif-H genes showed that the majority of the isolates (87%) showed 99% sequence homology with Rhizobium spp. obtained from NCBI gene data base and a few isolates (AAUGR-2, AAUGR-24) were grouped into Ensifer meliloti and Rhizobium leguminosarum var viceae indicating that grass pea rhizobia were more diverse than the hitherto established cross-nodulation grouping to Rhizobium leguminosarum var viceae. All the selected rhizobial isolates were highly significant (P=0.000) in their ability to solubilize TCP with zones of solubilisation (solubilization index; SI) ranging from 1.24- 3.37 cm and the capacity of producing organic acids within the range of 256- 418 μg/ml. The highest amount of IAA (74.69±1.72 μg/ml) was produced by Rhizobium species (AAUGR-14) followed by 74.18±1.95 μg/ml produced by AAUGR-30. Regarding the antagonistic properties, all isolates produced catalase and ammonia whereas, a number of isolates were positive for chitinase, protease, cellulase, HCN and in vitro fungal inhibition assay. Among a total of 100 isolates of rhizobacteria, 39% were phosphate solubilizers of which 22 isolates that showed SI of greater than 2.4 cm were selected for further screening process for multiple growth promoting properties (MPGP). The 16S rRNA gene sequence analysis of five rhizobacterial isolates, AAUGPR- 38, 53, 73, 91 and 92 showed 94- 99 % identity to different rhizobacterial genera including Enterobacter, Enterococcus, Kluyvera, pantoea and Serratia type. Under the circumstances most of them displayed 5-8 MPGP of which AAUGPR-53 identified as Enterococcus species, Enterococcus casseliflavus strain LAHHAB-24 and Enterococcus gallinarum strain F1 showed the highest phosphate solubilization index (PSI) and IAA production efficiency of 4.81±0.02 (cm) and 56.55±0.45 (μg/ml), respectively. Seventeen (77.3%) of the isolates showed in vitro antifungal inhibition against Fusarium oxysporum f. sp. lentis with isolates AAUGPR- 92 and 91 identified as Enterococcus species, Enterococcus casseliflavus and Enterococcus gallinarum exhibiting a percent radial growth inhibition of 73 and 83%, respectively. In general, 46%-100% of the isolates showed variations in their ability to produce chitinase, protease, cellulase, HCN and NH3 associated with antagonistic properties of the isolates. With regard to the performance of the selected inoculants to inoculation and coinoculation result, Rhizobium. sp. 14 performed xvi best in all single and co-inoculation trials with E. casseliflavus/gallinarum- 92. In general, the mixed inoculants; Rhizobium sp. 6, R. sp. 9, R. sp. 11, R. sp. 14 and E. casseliflavus/gallinarum-53 & 92 displayed the highest performance in all parameters. In all cases, treatments increased nodulation, growth and symbiotic effectiveness over the negative and positive controls. The mixed inoculants can be recommended as good candidates for plant growth improvement trials and validation under field conditions to exploit the crop for its multipurpose use with higher tolerance under extreme environmental factors, higher protein content and high yield under low soil fertility/input in the country.



Co-Inoculation, Rhizobacteria, Rhizobium Spp, Rhizobium Leguminosarum, Stress Tolerance