Fassil Assefa

Phylogeny and genetic diversity of native rhizobia nodulating common bean (Phaseolus vulgaris L.) in Ethiopia.

The diversity and phylogeny of 32 rhizobial strains isolated from nodules of common bean plants grown on 30 sites in Ethiopia were examined using AFLP fingerprinting and MLSA. Based on cluster analysis of AFLP fingerprints, test strains were grouped into six genomic clusters and six single positions. In a tree built from concatenated sequences of recA, glnII, rpoB and partial 16S rRNA genes, the strains were distributed into seven monophyletic groups. The strains in the groups B, D, E, G1 and G2 could be classified as Rhizobium phaseoli, R. etli, R. giardinii, Agrobacterium tumefaciens complex and A. radiobacter, respectively, whereas the strains in group C appeared to represent a novel species. R. phaseoli, R. etli, and the novel group were the major bean nodulating rhizobia in Ethiopia. The strains in group A were linked to R. leguminosarum species lineages but not resolved. Based on recA, rpoB and 16S rRNA genes sequences analysis, a single test strain was assigned as R. leucaenae. In the nodC tree the strains belonging to the major nodulating groups were clustered into two closely linked clades. They also had almost identical nifH gene sequences. The phylogenies of nodC and nifH genes of the strains belonging to R. leguminosarum, R. phaseoli, R. etli and the putative new species (collectively called R. leguminosarum species complex) were not consistent with the housekeeping genes, suggesting symbiotic genes have a common origin which is different from the core genome of the species and indicative of horizontal gene transfer among these rhizobia.

Genetic Diversity and Population Structure of Ethiopian Chickpea (Cicer arietinum L.) Germplasm Accessions from Different Geographical Origins as Revealed by Microsatellite Markers

Genetic diversity and population structure were studied in 155 chickpea (Cicer arietinum L.) entries using 33 polymorphic microsatellite (SSR) markers. Molecular analysis of variance showed variations of 73% within and 27% among populations. Introduced genotypes exhibited highest polymorphism (70.27%) than the landraces (36–57%). Collections from Shewa, Harerge, W. Gojam and S. Gonder regions also showed higher polymorphism (50–57%) than the rest of the local accessions (36–45%). Analyses of pairwise population Nei genetic distance and PhiPT coefficients, expected heterozygosity (He) and unbiased expected heterozygosity (UHe), Shannon’s information index (I) and percent polymorphism (% P) showed existence of high genetic variation between geographical regions. Accessions from adjoining geographical regions mostly showed more genetic similarities than those from origins far isolated apart. This could be associated with the ease and likelihood of inter-regional gene flow and seed movement particularly during times of drought. The 155 entries were grouped into five clusters following analysis of population structure. The first cluster (C1) constituted accessions from Arsi; the second (C2) from Gojam and Gonder; the third (C3) from Harerge and E. and N. Shewa; and the fourth (C4) from W. Shewa, Tigray, and Wello regions. The fifth cluster (C5) was entirely consisted of improved genotypes. Improved genotypes of both Kabuli and Desi types distinctly fell into cluster five (C5) regardless of their difference in seed types. The result has firmly established that introduction of genetic materials from exotic sources has broadened the genetic base of the national chickpea breeding program. Further implications of the findings as regards to chickpea germplasm management and its utilization in breeding program are also discussed.

Coffee husk composting: An investigation of the process using molecular and non-molecular tools

Highlights • Addition of cow dung to coffee husks improved composting kinetics.• Use of manure and fruit/vegetable wastes led to higher losses in total carbon.• DGGE indicated distinctive bacterial and fungal community shifts during composting.• COMPOCHIP revealed differences in quality and species diversity among composts.

Microbial Community Structure and Diversity in an Integrated System of Anaerobic-Aerobic Reactors and a Constructed Wetland for the Treatment of Tannery Wastewater in Modjo, Ethiopia

A culture-independent approach was used to elucidate the microbial diversity and structure in the anaerobic-aerobic reactors integrated with a constructed wetland for the treatment of tannery wastewater in Modjo town, Ethiopia. The system has been running with removal efficiencies ranging from 94%–96% for COD, 91%–100% for SO42- and S2-, 92%–94% for BOD, 56%–82% for total Nitrogen and 2%–90% for NH3-N. 16S rRNA gene clone libraries were constructed and microbial community assemblies were determined by analysis of a total of 801 unique clone sequences from all the sites. Operational Taxonomic Unit (OTU) - based analysis of the sequences revealed highly diverse communities in each of the reactors and the constructed wetland. A total of 32 phylotypes were identified with the dominant members affiliated to Clostridia (33%), Betaproteobacteria (10%), Bacteroidia (10%), Deltaproteobacteria (9%) and Gammaproteobacteria (6%). Sequences affiliated to the class Clostridia were the most abundant across all sites. The 801 sequences were assigned to 255 OTUs, of which 3 OTUs were shared among the clone libraries from all sites. The shared OTUs comprised 80 sequences belonging to Clostridiales Family XIII Incertae Sedis, Bacteroidetes and unclassified bacterial group. Significantly different communities were harbored by the anaerobic, aerobic and rhizosphere sites of the constructed wetland. Numerous representative genera of the dominant bacterial classes obtained from the different sample sites of the integrated system have been implicated in the removal of various carbon- containing pollutants of natural and synthetic origins. To our knowledge, this is the first report of microbial community structure in tannery wastewater treatment plant from Ethiopia.

Characterization of rhizobacteria isolated from wild Coffea arabica L.

Rhizobacteria from wild Arabica coffee populations (Coffea arabica L.) in southwestern Ethiopia were isolated and characterized. The main purpose was to identify coffee‐associated rhizobacteria and evaluate their potential in synthesizing the phytohormone indole acetic acid (IAA) and in degrading the ethylene precursor 1‐aminocyclopropane‐1‐carboxylate (ACC). A total of 878 bacterial isolates were screened, of which 395 (45%) isolates were preliminarily characterized using metabolic identification kits (API). Both Gram‐negative and Gram‐positive bacteria were isolated, with the former group predominating (63% of cases). Based on pre‐screening results of the biochemical tests, 51 of the isolates were subjected to PCR‐RFLP (Restriction Fragment Length Polymorphism) analysis that yielded ten groups, of which 24 isolates were identified by 16S rRNA gene sequencing. The major genera identified were Pseudomonas (six species) and Bacillus (four species). Single species of Erwinia, Ochrobactrum and Serratia were also identified. The Erwinia sp., Serratia marcescens and many Pseudomonas spp. produced IAA, and some isolates (all Pseudomonas spp.) were also able to degrade ACC. Several of the microbes found in association with wild Arabica coffee bushes have potential agronomic importance, like e.g. Bacillus thuringiensis, which deserve further testing. According to these in vitro studies, isolates of Erwinia, Serratia and Pseudomonas are of particular interest in inoculant development due to their plant growth promoting traits.

Phenotypic diversity for symbio-agronomic characters in Ethiopian chickpea (Cicer arietinum L.) germplasm accessions

Breeding chickpea ( Cicer arietinum L.) cultivars combining desirable symbiotic and agronomic characters has both economic and ecological significance. An experiment was conducted at Ambo and Ginchi, Ethiopia, in 2009/10 to characterize and evaluate 155 genotypes of chickpea for symbiotic and agronomic performance. A randomized complete block design with four replications and the difference technique, with a genetically non-nodulating chickpea genotype as a reference crop were employed to estimate the amount of symbiotic nitrogen fixation. Data analysis of 32 agronomic and symbiotic characters showed significant differences among the genotypes for all traits under study. Trait-based cluster analysis grouped the genotypes into six different classes. Standardized Mahalanobis D 2 statistics showed significant genetic distances between all clusters constituted local landraces and introduced genotypes. This indicated that there were distinct multivariate differences between landraces and introduced genotypes. No clear interrelationship was observed between the geographic origins of the landraces and the pattern of genetic diversity, as there were accessions from the same source of origin that fell into different clusters and vice versa. Different symbiotic and agronomic characters had different contribution to the total differences among the populations. Those characters that contributed more to the total differentiation of the populations and genotypes into the different clusters should be exploited in future breeding. Keywords: Chickpea ( Cicer arietinum ), cluster analysis, genetic diversity, germplasm, symbiotic nitrogen fixation

Phosphate solubilization and multiple plant growth promoting properties of rhizobacteria isolated from chickpea ( Cicer aeritinum L.) producing areas of Ethiopia

Chickpea is one of the major legume crops widely grown in Ethiopia. The low availability of phosphorus in soil is among the stresses that constrain the production of this crop in the country. However, there are rhizobacteria capable of solubilizing insoluble forms of phosphorus in soil and make it available to the plant. Thus, this study was aimed at isolation and characterization of phosphate solubilizing bacteria from chickpea rhizosphere. Fifty phosphate solubilizing bacterial strains were isolated from the soil samples, characterized biochemically and identified by 16S rDNA sequences analysis. The results indicate the presence of genera Acinetobacter, Bacillus, Brevibacillus, Burkholderia, Empedobacter, Enterobacter, Pseudomonas, Ralstonia, Sphingomonas and Stenotrophomonas. Phosphate solubilizing efficiencies of the strains were analyzed using different insoluble phosphorus sources and the results show that most isolates released a substantial amount of soluble phosphate from tricalcium phosphate, rock phosphate and bone meal. Screening for multiple plant growth promoting attributes showed that 44 and 18% of them were capable of producing indole acetic acid and inhibiting the growth of Fusarium oxysporum under in vitro conditions, respectively. A direct impact of several strains (Bacillus flexus (PSBC17), Pseudomonas fluorescence (PSBC33), Enterobacter sp. (PSBC35), Enterobacter sakazaki (PSBC79) and Enterobacter sp. (PSBC81)) on the growth of chickpea in pot culture has been demonstrated by the increase in the number of root nodules, shoot dry matter, nitrogen and phosphorus concentration of shoot. Based on the results, we conclude that chickpea rhizosphere harbor phosphate solubilizing bacteria which are diverse in taxonomy and phosphate solubilizing efficiencies. Thus, consecutive studies should focus on field studies on those strains due to their potentially high importance for the phosphorus nutrition of crops in this area and in this context for the improvement of the sustainability of crop production in the country

The Role of Microbial Aspartic Protease Enzyme in Food and Beverage Industries

Proteases represent one of the three largest groups of industrial enzymes and account for about 60% of the total global enzymes sale. According to the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology, proteases are classified in enzymes of class 3, the hydrolases, and the subclass 3.4, the peptide hydrolases or peptidase. Proteases are generally grouped into two main classes based on their site of action, that is, exopeptidases and endopeptidases. Protease has also been grouped into four classes based on their catalytic action: aspartic, cysteine, metallo, and serine proteases. However, lately, three new systems have been defined: the threonine-based proteasome system, the glutamate-glutamine system of eqolisin, and the serine-glutamate-aspartate system of sedolisin. Aspartic proteases (EC 3.4.23) are peptidases that display various activities and specificities. It has two aspartic acid residues (Asp32 and Asp215) within their active site which are useful for their catalytic activity. Most of the aspartic proteases display best enzyme activity at low pH (pH 3 to 4) and have isoelectric points in the pH range of 3 to 4.5. They are inhibited by pepstatin. The failure of the plant and animal proteases to meet the present global enzyme demand has directed to an increasing interest in microbial proteases. Microbial proteases are preferred over plant protease because they have most of the characteristics required for their biotechnological applications. Aspartic proteases are found in molds and yeasts but rarely in bacteria. Aspartic protease enzymes from microbial sources are mainly categorized into two groups: (i) the pepsin-like enzymes produced by Aspergillus, Penicillium, Rhizopus, and Neurospora and (ii) the rennin-like enzymes produced by Endothia and Mucor spp., such as Mucor miehei, M. pusillus, and Endothia parasitica. Aspartic proteases of microbial origin have a wide range of application in food and beverage industries. These include as milk-clotting enzyme for cheese manufacturing, degradation of protein turbidity complex in fruit juices and alcoholic liquors, and modifying wheat gluten in bread by proteolysis.