Phytochemical profiling, toxicity studies, wound healing, analgesic and anti-inflammatory activities of Musa paradisiaca L. Musaceae (Plantain) stem extract in rats.

ETHNOPHARMACOLOGICAL RELEVANCE: The stem of Musa paradisiaca (plantain) has found application in traditional medicine for the treatment of diabetes, inflammation, ulcers and wound injuries. AIM OF THE STUDY: This study investigated the phytochemical composition, toxicity profile, wound healing, anti-inflammatory and analgesic effects of aqueous Musa paradisiaca stem extract (AMPSE) in rats. METHODS: Phytochemical analysis of methanol-MPSE was performed by gas chromatography-mass spectrometry (GC-MS). Acute toxicity testing was carried out through oral administration of a single dose of AMPSE up to 5 g/kg. Four separate groups of rats were used for the subacute toxicity testing (n = 6). Group 1 served as a normal control and did not receive AMPSE, groups 2-4 received AMPSE daily by gavage for 28 days. In the experiments with excision and incision wounds, the rats were treated with 10 w/w AMPS extract. The anti-inflammatory and analgesic effects of AMPSE were assessed using egg albumin-induced paw oedema and acetic acid-induced writhing methods, respectively. For the subacute, anti-inflammatory and analgesic studies, AMPSE was administered to the experimental rats at doses of 300, 600 and 900 mg/kg body weight. RESULTS: Bioactive compounds identified include ß-sitisterol, n-hexadecanoic acid, octadecanoic acid, diethyl sulfate, p-hydroxynorephedrine, phenylephrine, nor-pseudoephedrine, metaraminol, pseudoephedrine and vanillic acid. No signs of toxicity and no deaths were observed in all the groups. For the groups treated with AMPSE for 28 days, a significant reduction in alkaline phosphatase, alanine aminotransferase, aspartate aminotransferase, urea, sodium, chloride, total cholesterol, triglycerides, and low-density lipoprotein cholesterol were observed while high density lipoprotein cholesterol, glutathione and superoxide dismutase increased compared to control (p < 0.05). In wound healing experiments, AMPSE showed greater percent wound contraction and wound resistance fracture compared to the povidone-iodine (PI) treated and control groups. Treatment with 900 mg/kg AMPSE resulted in significant (p < 0.05) anti-inflammatory and analgesic effects compared to the control. CONCLUSION: This study shows that AMPSE is not toxic but contains biologically active compounds with hepatoprotective, anti-inflammatory, lipid-lowering and wound-healing effects. Treatment of rats with AMPSE has shown that AMPSE has anti-inflammatory, analgesic, hepatoprotective, lipid-lowering and wound-healing effects, supporting its therapeutic use in ethnomedicine.

Diarylheptanoid Derivatives (Musellins A-F) and Dimeric Phenylphenalenones from Seed Coats of Musella lasiocarpa, the Chinese Dwarf Banana.

Phenylphenalenones (PPs) are phytoalexins protecting banana plants (Musaceae) against various pathogens. However, how plants synthesize PPs is still poorly understood. In this work, we investigated the major secondary metabolites of developing seed coats of Musella lasiocarpa to determine if this species might be a good model system to study the biosynthesis of PPs. We found that PPs are major components of M. lasiocarpa seed coats at middle and late developmental stages. Two previously undescribed PP dimers (M-4 and M-6) and a group of unreported diarylheptanoid (DH) derivatives named musellins A-F (B-7, B-9, B-10, B-12, B-14, and B-15) were isolated along with 14 known compounds. Musellin D (B-12) and musellin F (B-15) contain the first reported furo[3,2-c]pyran ring and represent a previously undescribed carbon skeleton. The chemical structures of all new compounds were characterized by spectroscopic data, including NMR, HRESIMS, and ECD analysis. Plausible biosynthetic pathways for the formation of PPs and DHs are proposed.

On-farm diversity, use pattern, and conservation of enset (Ensete ventricosum) genetic resources in southern Ethiopia.

BACKGROUND: Enset is an important source of food and is consumed by about 25 million people as a staple or co-staple food crop mainly in southern parts of Ethiopia. Large numbers of enset landraces exist in different administrative zones of Ethiopia with a wide range of altitudes and agroclimatic zones. However, limited information is available on the diversity, distribution, and utilization pattern corresponding to the diverse ethnolinguistic as well as sociocultural communities of the country. Hence, this study was devised to explore and document the richness of farmers' tradition and practice on the diversity and distribution of enset landraces on the farm level and selection pattern for different purposes regarding the production, utilization, and conservation of enset genetic resources. METHODS: The study was conducted in four major enset-growing administrative zones of Ethiopia, namely Hadiya, Kembata-Tembaro, Gurage, and Silte. A total of 240 farm households were surveyed using individual interviews, 18 key informant interviews, 36 focus group discussions with 5 participants, and direct on-farm field observations for data collection. Considering that enset has a rich cultural background and indigenous knowledge, ethnobotanical research approach was applied to data collection and analysis. The Shannon-Weaver, Simpson, Pielou, and Jaccard's similarity indices were used to evaluate the diversity and similarity of the landraces as well as using descriptive statistics in SPSS Ver. 24. Preference in direct matrix ranking was also used to compute and rank the enset landraces most preferred by the people in the context of specific use value in the study area. RESULTS: A total of 282 farmer-named enset landraces have been identified, with a range from 2 to 32 on individual homegardens. The largest number of landraces was found in the Hadiya Zone (86), while the lowest was scored in the Silte Zone (57). The Shannon diversity index (H') ranged from 3.73 (Silte) to 3.96 (Hadiya). Similarly, landraces revealed a very narrow range of variances in Simpson's 1-D diversity index, and it ranged from 0.963 (Silte) to 0.978 (Hadiya). Likewise, the similarity index ranged from 0.24 to 0.73 sharing 16-47 landraces in common. Of the 282 landraces, 210 (74.5%) were recorded in more than one zones, whereas 72 (25.5%) had narrow distribution being restricted to a single zone. CONCLUSIONS: Farmers have established long-term practices and experiences in cultivation, utilization, and conservation of a diverse group of enset landraces to fill their domestic and market purposes in each zone. The variation is likely to be related to agroclimatic differences, ethnicity factors, food cultures, and historical backgrounds. Therefore, to facilitate on-farm conservation as well as sustainable utilization of the enset genetic resources, farmers need to be supported by different stakeholders for all their worth and also in crop improvement programs.

Dietary practices and nutritional status of young children in the former ensete monoculture dominated Sidama region, southern Ethiopia: A community based cross-sectional study.

BACKGROUND: Child undernutrition is a challenge in Ethiopia, where morbidity and mortality among children are attributed to it. This study aimed to describe the dietary practices, household food insecurity, and nutritional status of young children in Dale district, Sidama region, southern Ethiopia. We discuss our findings in light of research from the same area 3 to 5 decades ago, and we analyze factors associated with linear growth of young children. METHOD: A community-based cross-sectional study design was employed. Children less than two years old and their primary caretakers (n = 903) were included in this study. Among whom 791 children were aged above six months and 742 children out of 791 provided a 24-hour dietary recall. Interviews capturing other dietary practices, food insecurity, socio-demographic characteristics, anthropometric measurements, and haemoglobin assessments were performed for all. The WHO Child Growth Standards were used to calculate anthropometric indices and to describe stunting (length-for-age z-score <-2). Haemoglobin measures below 11g/dl were defined as low haemoglobin levels (anaemia). Multilevel linear regression was used to identify factors associated with length-for-age z-scores. RESULT: The prevalence of stunting, wasting, and anaemia was 39.5%, 3.9%, and 61.7%, respectively. The majority of children (97%) ate cereals (maize) during the past 24 hours, and 79.6% of households use maize as the main food. Three fourth (75%) of the total households were food insecure ranging from mild to severe food insecurity. Boys were at higher risk of having lower length-for-age z-score than girls (ß -0.53; 95% Confidence Interval (CI): -0.78, -0.27). For each month the children grew older, length-for-age z-score decreased slightly (ß -0.06; -0.07, -0.04). Household food insecurity scores (ß -0.05; -0.08, -0.01) and children haemoglobin levels, (ß 0.21; 0.06, 0.35) were also associated with length-for-age z-score among young children. CONCLUSION: Linear growth failure (stunting) was prevalent among young children in the rural Sidama region and the majority of them were also anaemic. Older age, male sex, a lower haemoglobin level in children, and household food insecurity were risk factors of linear growth failure in young children. Maize seems to be the dominant food in this previously ensete dominated area; however, there have been minimal improvements in length-for-age z-score over decades. Strategies to ensure household's food access and improve the quality of child diets need to be implemented.

Genome wide identification of MADS box gene family in Musa balbisiana and their divergence during evolution.

MADS box gene family is transcription factor gene family that is involved in growth and development of eukaryotes. In plants the MADS box gene family is mainly associated with floral meristem identity and flower development, apart from being involved in nearly all the phases of plant growth. The MADS box gene family has also been shown to be involved during fruit development and ripening. In this study the MADS box gene family from Musa balbisiana was identified and the divergence of this gene family between Musa balbisiana and Musa acuminata studied. A total of 97 MADS box genes were identified from the genome of Musa balbisiana. Phylogenetic analysis showed that the MbMADS box genes were categorised into type I (α and γ; the ß group was not distinguishable) and type II groups (MIKCc and MIKC* and MIKCc was further divided into 13 subfamilies). The typeII group has the largest number of genes and also showed the most expansion which could be correlated with the whole genome duplications. There were significant differences in the MADS box genes from Musa acuminata and Musa balbisiana during evolution that can be correlated with different floral phenotype and fruit ripening pattern. The divergence of the MADS RIN genes in Musa balbisiana as compared to Musa acuminata might play an important role in the slow ripening of Musa balbisiana fruits.

A chromosome-level reference genome of Ensete glaucum gives insight into diversity and chromosomal and repetitive sequence evolution in the Musaceae.

BACKGROUND: Ensete glaucum (2n = 2x = 18) is a giant herbaceous monocotyledonous plant in the small Musaceae family along with banana (Musa). A high-quality reference genome sequence assembly of E. glaucum is a resource for functional and evolutionary studies of Ensete, Musaceae, and the Zingiberales. FINDINGS: Using Oxford Nanopore Technologies, chromosome conformation capture (Hi-C), Illumina and RNA survey sequence, supported by molecular cytogenetics, we report a high-quality 481.5 Mb genome assembly with 9 pseudo-chromosomes and 36,836 genes. A total of 55% of the genome is composed of repetitive sequences with predominantly LTR-retroelements (37%) and DNA transposons (7%). The single 5S ribosomal DNA locus had an exceptionally long monomer length of 1,056 bp, more than twice that of the monomers at multiple loci in Musa. A tandemly repeated satellite (1.1% of the genome, with no similar sequence in Musa) was present around all centromeres, together with a few copies of a long interspersed nuclear element (LINE) retroelement. The assembly enabled us to characterize in detail the chromosomal rearrangements occurring between E. glaucum and the x = 11 species of Musa. One E. glaucum chromosome has the same gene content as Musa acuminata, while others show multiple, complex, but clearly defined evolutionary rearrangements in the change between x= 9 and 11. CONCLUSIONS: The advance towards a Musaceae pangenome including E. glaucum, tolerant of extreme environments, makes a complete set of gene alleles, copy number variation, and a reference for structural variation available for crop breeding and understanding environmental responses. The chromosome-scale genome assembly shows the nature of chromosomal fusion and translocation events during speciation, and features of rapid repetitive DNA change in terms of copy number, sequence, and genomic location, critical to understanding its role in diversity and evolution.

Comparative plastome analysis of Musaceae and new insights into phylogenetic relationships.

BACKGROUND: Musaceae is an economically important family consisting of 70-80 species. Elucidation of the interspecific relationships of this family is essential for a more efficient conservation and utilization of genetic resources for banana improvement. However, the scarcity of herbarium specimens and quality molecular markers have limited our understanding of the phylogenetic relationships in wild species of Musaceae. Aiming at improving the phylogenetic resolution of Musaceae, we analyzed a comprehensive set of 49 plastomes for 48 species/subspecies representing all three genera of this family. RESULTS: Musaceae plastomes have a relatively well-conserved genomic size and gene content, with a full length ranging from 166,782 bp to 172,514 bp. Variations in the IR borders were found to show phylogenetic signals to a certain extent in Musa. Codon usage bias analysis showed different preferences for the same codon between species and three genera and a common preference for A/T-ending codons. Among the two genes detected under positive selection (dN/dS > 1), ycf2 was indicated under an intensive positive selection. The divergent hotspot analysis allowed the identification of four regions (ndhF-trnL, ndhF, matK-rps16, and accD) as specific DNA barcodes for Musaceae species. Bayesian and maximum likelihood phylogenetic analyses using full plastome resulted in nearly identical tree topologies with highly supported relationships between species. The monospecies genus Musella is sister to Ensete, and the genus Musa was divided into two large clades, which corresponded well to the basic number of n = x = 11 and n = x =10/9/7, respectively. Four subclades were divided within the genus Musa. A dating analysis covering the whole Zingiberales indicated that the divergence of Musaceae family originated in the Palaeocene (59.19 Ma), and the genus Musa diverged into two clades in the Eocene (50.70 Ma) and then started to diversify from the late Oligocene (29.92 Ma) to the late Miocene. Two lineages (Rhodochlamys and Australimusa) radiated recently in the Pliocene /Pleistocene periods. CONCLUSIONS: The plastome sequences performed well in resolving the phylogenetic relationships of Musaceae and generated new insights into its evolution. Plastome sequences provided valuable resources for population genetics and phylogenetics at lower taxon.

Reproductive biology of wild and domesticated Ensete ventricosum: Further evidence for maintenance of sexual reproductive capacity in a vegetatively propagated perennial crop.

Loss of sexual reproductive capacity has been proposed as a syndrome of domestication in vegetatively propagated crops, but there are relatively few examples from agricultural systems. In this study, we compare sexual reproductive capacity in wild (sexual) and domesticated (vegetative) populations of enset (Ensete ventricosum (Welw.) Cheesman), a tropical banana relative and Ethiopian food security crop. We examined floral and seed morphology and germination ecology across 35 wild and domesticated enset. We surveyed variation in floral and seed traits, including seed weight, viability and internal morphology, and germinated seeds across a range of constant and alternating temperature regimes to characterize optimum germination requirements. We report highly consistent floral allometry, seed viability, internal morphology and days to germination in wild and domesticated enset. However, seeds from domesticated plants responded to cooler temperatures with greater diurnal range. Shifts in germination behaviour appear concordant with a climatic envelope shift in the domesticated distribution. Our findings provide evidence that sexual reproductive capacity has been maintained despite long-term near-exclusive vegetative propagation in domesticated enset. Furthermore, certain traits such as germination behaviour and floral morphology may be under continued selection, presumably through rare sexually reproductive events. Compared to sexually propagated crops banked as seeds, vegetative crop diversity is typically conserved in living collections that are more costly and insecure. Improved understanding of sexual propagation in vegetative crops may have applications in germplasm conservation and plant breeding.

Genome assembly of Musa beccarii shows extensive chromosomal rearrangements and genome expansion during evolution of Musaceae genomes.

BACKGROUND: Musa beccarii (Musaceae) is a banana species native to Borneo, sometimes grown as an ornamental plant. The basic chromosome number of Musa species is x = 7, 10, or 11; however, M. beccarii has a basic chromosome number of x = 9 (2n = 2x = 18), which is the same basic chromosome number of species in the sister genera Ensete and Musella. Musa beccarii is in the section Callimusa, which is sister to the section Musa. We generated a high-quality chromosome-scale genome assembly of M. beccarii to better understand the evolution and diversity of genomes within the family Musaceae. FINDINGS: The M. beccarii genome was assembled by long-read and Hi-C sequencing, and genes were annotated using both long Iso-seq and short RNA-seq reads. The size of M. beccarii was the largest among all known Musaceae assemblies (∼570 Mbp) due to the expansion of transposable elements and increased 45S ribosomal DNA sites. By synteny analysis, we detected extensive genome-wide chromosome fusions and fissions between M. beccarii and the other Musa and Ensete species, far beyond those expected from differences in chromosome number. Within Musaceae, M. beccarii showed a reduced number of terpenoid synthase genes, which are related to chemical defense, and enrichment in lipid metabolism genes linked to the physical defense of the cell wall. Furthermore, type III polyketide synthase was the most abundant biosynthetic gene cluster (BGC) in M. beccarii. BGCs were not conserved in Musaceae genomes. CONCLUSIONS: The genome assembly of M. beccarii is the first chromosome-scale genome assembly in the Callimusa section in Musa, which provides an important genetic resource that aids our understanding of the evolution of Musaceae genomes and enhances our knowledge of the pangenome.

Microbial community shifts association with physicochemical parameters: Visualizing enset bacterial wilt from different states of enset health.

Bacterial wilt of enset caused by Xanthomonas campestris is a devastating disease in Ethiopia, where enset is domesticated and served as a staple food for about 20 million people in the country. While enset is infected by bacteria, it shows different wilting stages. However, the microbial community shifts at the different stages of enset infection and associated physicochemical parameter changes remain poorly understood. This study was aimed to visualize the proportion of enset wilt bacterium from other microbial community and its association with physicochemical parameter at different states of enset health. Soil and enset (zero, first, second and third stages) samples were collected from three districts in Gamo Highlands for physicochemical and biological (culture dependent and16S rRNA gene sequence) analysis. The results of culture dependent analysis which has been complemented by 16S rRNA gene sequence confirmed that increasing trends were observed for Xanthomonadaceae, Pseudomonadaceae, Lactobacillaceae and Flavobacteriaceae, while Bacillaceae and Enterobacteriaceae showed progressive decrease from zero to the third stage. Particularly, the 16S rRNA data showed that Xanthomonadaceae increased significantly from zero to different (2.5 × 102 times at the onset of disease and 1.0-2.0 × 104 times at the second and third) stages of enset infection. Most physicochemical results showed that a decreasing trends from zero to third stage, while few parameters are showing an increasing trend. Moisture content (R2 ≥ 0.951, P ≤ 0.049) of the soil and plant samples positively influenced Xanthomonas abundance, while this bacterium showed a strongly negative significant correlation with pH (R2 ≥ -0.962, P ≤ 0.038), temperature (R2 ≥ -0.958, P ≤ 0.042), OM (R2 ≥ -0.952, P ≤ 0.048), and TN (R2 ≥ -0.951, P ≤ 0.049). A strongly negative significant correlation (R2 ≥ -0.948, P ≤ 0.050) was also observed between Xanthomonas and nutrients (K, Mg, Ca, and Cu). Overall, this study implies that different environmental factors found a key driving force of Xanthomonas proportional increment from low abundance at zero stage to higher abundance at the last stage of enset infection suggesting that considering these factors help to design an effective enset disease management strategy, for which further studies will be needed.

The pangenome of banana highlights differences between genera and genomes.

Banana (Musaceae family) has a complex genetic history and includes a genus Musa with a variety of cultivated clones with edible fruits, Ensete species that are grown for their edible corm, and monospecific Musella whose generic status has been questioned. The most commonly exported banana cultivars belong to Cavendish, a subgroup of Musa triploid cultivars, which is under threat by fungal pathogens, though there are also related species M. balbisiana Colla (B genome), M. textilis Née (T genome), and M. schizocarpa N. W. Simmonds (S genome), along with hybrids of these genomes, which potentially host genes of agronomic interest. Here we present the first cross-genus pangenome of banana, which contains representatives of the Musa and Ensete genera. Clusters based on gene presence-absence variation (PAV) clearly separate Musa and Ensete, while Musa is split further based on species. These results present the first pangenome study across genus boundaries and identifies genes that differentiate between Musaceae species, information that may support breeding programs in these crops.

Micronutrient composition and microbial community analysis across diverse landraces of the Ethiopian orphan crop enset.

Enset (Ensete ventricosum) is a major starch staple and food security crop for 20 million people. Despite substantial diversity in morphology, genetics, agronomy and utilization across its range, nutritional characteristics have only been reported in relatively few landraces. Here, we survey nutritional composition in 22 landraces from three enset growing regions. We present mineral characterization of enset corm tissue, free amino acid characterization of raw and processed (fermented) tissues and genomic analysis of the microbial community associated with fermentation. We show that compared to regionally important tubers and cereals, enset is high in calcium, iron, potassium and zinc and low in sodium. We report changes in free amino acid composition due to processing, and establish that the bacteria genera Acetobacter, Lactobacillus and Bifidobacterium, predominate during fermentation. Nutritional and microbial variation presents opportunities to select for improved composition, quality and safety with potentially significant impacts in food security and public health.

The landscape of microsatellites in the enset (Ensete ventricosum) genome and web-based marker resource development.

Ensete ventricosum (Musaceae, enset) is an Ethiopian food security crop. To realize the potential of enset for rural livelihoods, further knowledge of enset diversity, genetics and genomics is required to support breeding programs and conservation. This study was conducted to explore the enset genome to develop molecular markers, genomics resources, and characterize enset landraces while giving insight into the organization of the genome. We identified 233 microsatellites (simple sequence repeats, SSRs) per Mbp in the enset genome, representing 0.28% of the genome. Mono- and di-nucleotide repeats motifs were found in a higher proportion than other classes of SSR-motifs. In total, 154,586 non-redundant enset microsatellite markers (EMM) were identified and 40 selected for primer development. Marker validation by PCR and low-cost agarose gel electrophoresis revealed that 92.5% were polymorphic, showing a high PIC (Polymorphism Information Content; 0.87) and expected heterozygosity (He = 0.79-0.82). In silico analysis of genomes of closely related species showed 46.86% of the markers were transferable among enset species and 1.90% were transferable to Musa. The SSRs are robust (with basic PCR methods and agarose gel electrophoresis), informative, and applicable in measuring enset diversity, genotyping, selection and potentially breeding. Enset SSRs are available in a web-based database at https://enset-project.org/EnMom@base.html (or https://enset.aau.edu.et/index.html , downloadable from Figshare).

Ensete ventricosum: A Multipurpose Crop against Hunger in Ethiopia.

Ensete ventricosum is a traditional multipurpose crop mainly used as a staple/co-staple food for over 20 million people in Ethiopia. Despite this, scientific information about the crop is scarce. Three types of food, viz., Kocho (fermented product from scraped pseudostem and grated corm), Bulla (dehydrated juice), and Amicho (boiled corm) can be prepared from enset. These products are particularly rich in carbohydrates, minerals, fibres, and phenolics, but poor in proteins. Such meals are usually served with meat and cheese to supplement proteins. As a food crop, it has useful attributes such as foods can be stored for long time, grows in wide range of environments, produces high yield per unit area, and tolerates drought. It has an irreplaceable role as a feed for animals. Enset starch is found to have higher or comparable quality to potato and maize starch and widely used as a tablet binder and disintegrant and also in pharmaceutical gelling, drug loading, and release processes. Moreover, enset shows high genetic diversity within a population which in turn renders resilience and food security against the ever-changing environmental factors and land use dynamics. Therefore, more research attention and funding should be given to magnify and make wider use of the crop.

Preparation, Optimization, and Evaluation of Epichlorohydrin Cross-Linked Enset (Ensete ventricosum (Welw.) Cheeseman) Starch as Drug Release Sustaining Excipient in Microsphere Formulation.

Ensete ventricosum (Welw.) cheeseman which belongs to the family of Musaceae is one of the main sources of starch in Ethiopia. This study aimed at evaluating epichlorohydrin cross-linked enset starch as a drug release sustaining excipient in microsphere formulations of theophylline. Extracted enset starch was cross-linked using epichlorohydrin as a cross-linking agent. The effect of cross-linker concentration, cross-linking duration, and cross-linking temperature on the degree of cross-linking and release rate of microspheres prepared by emulsion solvent evaporation method was investigated using the two-level full factorial design. Accordingly, the concentration of epichlorohydrin and duration of cross-linking were the most significant factors affecting both the degree of cross-linking and drug release rate. Thus, the effects of these two factors were further studied and optimized using the central composite design. As per the numerical method of central composite design, the optimal points were obtained at epichlorohydrin concentration of 13.70% and cross-linking time of 3.82 h. Under these optimal conditions, the model predicts the degree of cross-linking of 74.70% and drug release rate of 28.00 h1/2. The validity of these optimal points was confirmed experimentally. The microspheres of the optimum formulation also exhibited minimum burst release with sustained release for 12 h. Besides, the optimized formulation followed the Higuchi square root kinetic model with non-Fickian diffusion release mechanism. The finding of this study suggested that cross-linked enset starch can be used as an alternative drug-release-sustaining pharmaceutical excipient in microsphere formulation.

Endophytic fungal diversity isolated from different agro-ecosystem of Enset (Ensete ventericosum) in Gedeo zone, SNNPRS, Ethiopia.

BACKGROUND: Endophytic Fungi (EF) are an underexplored group of microorganisms as only a few plants have been studied with regards to their community. Diversity of EF found in young and old leaves of Enset plant has not been well studied. We analyzed and compared the colonization frequency (CF), richness diversity and fungal communities of the EF inhabiting the young and old leaves of Enset plant from Southern region of Ethiopia. Standard methods were used for isolation and identification of endophytic species from Enset leaves. RESULTS: The study investigates the difference in quantity, in variety, in consistent pattern of community of EF along with different Enset varieties. A total number of 18 samples were analyzed and 108 morphospecies of EF were isolated and distributed among 17 genera. Aspergillus sp. and Penicillium sp. were the most common fungi reported in Enset plant. The largest numbers of EF isolates were observed in Maziya and Arkiya variety and the diversity index and species richness were found to be significant in Enset plant among these varieties. A high number of EF was isolated from old leaves in comparison to young leaves among all the varieties studied. Composition of EF at different altitudinal location also varied within each sites. CONCLUSION: Isolation, characterization and distribution of the EF from Enset plant is the first approach that has been conducted in the developing country like Ethiopia. The findings of the present study show that the Enset agro-forestry system produces potential variability in the colonization, richness diversity and composition of EF in Enset plants. The assemblage of EF in healthy tissues of Enset plants may indicate that some of the fungi are possible latent pathogens and some may become saprophytic.

A new Multi Locus Variable Number of Tandem Repeat Analysis Scheme for epidemiological surveillance of Xanthomonas vasicola pv. musacearum, the plant pathogen causing bacterial wilt on banana and enset.

Xanthomonas vasicola pv. musacearum (Xvm) which causes Xanthomonas wilt (XW) on banana (Musa accuminata x balbisiana) and enset (Ensete ventricosum), is closely related to the species Xanthomonas vasicola that contains the pathovars vasculorum (Xvv) and holcicola (Xvh), respectively pathogenic to sugarcane and sorghum. Xvm is considered a monomorphic bacterium whose intra-pathovar diversity remains poorly understood. With the sudden emergence of Xvm within east and central Africa coupled with the unknown origin of one of the two sublineages suggested for Xvm, attention has shifted to adapting technologies that focus on identifying the origin and distribution of the genetic diversity within this pathogen. Although microbiological and conventional molecular diagnostics have been useful in pathogen identification. Recent advances have ushered in an era of genomic epidemiology that aids in characterizing monomorphic pathogens. To unravel the origin and pathways of the recent emergence of XW in Eastern and Central Africa, there was a need for a genotyping tool adapted for molecular epidemiology. Multi-Locus Variable Number of Tandem Repeat Analysis (MLVA) is able to resolve the evolutionary patterns and invasion routes of a pathogen. In this study, we identified microsatellite loci from nine published Xvm genome sequences. Of the 36 detected microsatellite loci, 21 were selected for primer design and 19 determined to be highly typeable, specific, reproducible and polymorphic with two- to four- alleles per locus on a sub-collection. The 19 markers were multiplexed and applied to genotype 335 Xvm strains isolated from seven countries over several years. The microsatellite markers grouped the Xvm collection into three clusters; with two similar to the SNP-based sublineages 1 and 2 and a new cluster 3, revealing an unknown diversity in Ethiopia. Five of the 19 markers had alleles present in both Xvm and Xanthomonas vasicola pathovars holcicola and vasculorum, supporting the phylogenetic closeliness of these three pathovars. Thank to the public availability of the haplotypes on the MLVABank database, this highly reliable and polymorphic genotyping tool can be further used in a transnational surveillance network to monitor the spread and evolution of XW throughout Africa.. It will inform and guide management of Xvm both in banana-based and enset-based cropping systems. Due to the suitability of MLVA-19 markers for population genetic analyses, this genotyping tool will also be used in future microevolution studies.

Enset in Ethiopia: a poorly characterized but resilient starch staple.

BACKGROUND: Enset (Ensete ventricosum, Musaceae) is an African crop that currently provides the staple food for approx. 20 million Ethiopians. Whilst wild enset grows over much of East and Southern Africa and the genus extends across Asia to China, it has only ever been domesticated in the Ethiopian Highlands. Here, smallholder farmers cultivate hundreds of landraces across diverse climatic and agroecological systems. SCOPE: Enset has several important food security traits. It grows over a relatively wide range of conditions, is somewhat drought-tolerant, and can be harvested at any time of the year, over several years. It provides an important dietary starch source, as well as fibres, medicines, animal fodder, roofing and packaging. It stabilizes soils and microclimates and has significant cultural importance. In contrast to the other cultivated species in the family Musaceae (banana), enset has received relatively little research attention. Here, we review and critically evaluate existing research, outline available genomic and germplasm resources, aspects of pathology, and explore avenues for crop development. CONCLUSION: Enset is an underexploited starch crop with significant potential in Ethiopia and beyond. Research is lacking in several key areas: empirical studies on the efficacy of current agronomic practices, the genetic diversity of landraces, approaches to systematic breeding, characterization of existing and emerging diseases, adaptability to new ranges and land-use change, the projected impact of climate change, conservation of crop wild relatives, by-products or co-products or non-starch uses, and the enset microbiome. We also highlight the limited availability of enset germplasm in living collections and seedbanks, and the lack of knowledge of reproductive and germination biology needed to underpin future breeding. By reviewing the current state of the art in enset research and identifying gaps and opportunities, we hope to catalyse the development and sustainable exploitation of this neglected starch crop.

Effect of fermentation system on the physicochemical and microbial community dynamics during enset (Ensete ventricosum) fermentation.

AIMS: The present study was conducted to assess the effect of three different fermentation systems on fermentation of enset into kocho. METHODS AND RESULTS: Nine enset plants were processed, mixed and fermented in either a pit, a bamboo basket or a sauerkraut jar. Samples were taken on days 1, 7, 15, 31, 60 and 90. Moisture content and pH generally decreased and titratable acidity increased during fermentation. Total viable aerobic counts were generally high for all samples and Enterobacteriaceae counts were reduced to below the detectable level after day 1 for the pits and jars and after day 7 for the baskets. Illumina MiSeq sequencing of 16S ribosomal RNA genes revealed that Leuconostoc and Lactococcus spp. were the most abundant lactic acid bacteria in the initial phases of the fermentation. Later on, Lactobacillus, Weissella and Bifidobacterium dominated. CONCLUSIONS: The type of fermentation system used had an effect on the microbial dynamics and the effect increased towards the end of fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: Millions of people in Ethiopia daily consume kocho prepared in either a pit or a basket. These systems show practical problems, but this study shows that fermentation is also possible in a sauerkraut jar.

Yeasts and bacteria associated with kocho, an Ethiopian fermented food produced from enset (Ensete ventricosum).

Enset (Ensete ventricosum) is the basis of the staple food consumed by about 20% of the Ethiopian population. Kocho is one of the food products generated from enset by spontaneous fermentation of decorticated and pulverized pseudostem and corm sections. We isolated culturable microbes associated with kocho from different stages of fermentation. Twelve yeast species, six lactic acid bacteria (LABs) species and eleven species of aerobic bacteria were identified by sequencing ITS/D1D2 regions of 26S rDNA of yeasts and 16S rDNA of bacteria, respectively. More yeast species were identified in fresh (fermented for 2-5 days) kocho, compared to long-term (7-12 months) fermented kocho, while we observed an opposite trend for LABs. In fresh kocho, the most frequently isolated yeast species were Pichia exigua, Galactomyces geotrichum, and Pichia fermentans. From mid-term (3-4 months) kocho most frequently Candida cabralensis, G. geotrichum, and Candida ethanolica were isolated. In the long-term fermentations, the most frequently isolated yeast was Saturnispora silva. Lactobacillus plantarum was the most frequently isolated LAB in both fresh and mid-term kocho. In long-term fermented kocho, Acetobacter pasteurianus and L. plantarum were most frequently isolated. L. plantarum was consistently isolated from all the three stages of fermentation. Aerobic bacteria in fresh kocho were mostly gram-negative, with Raoultella planticola and Pantoea agglomerans being the most frequently isolated species. In long-term fermented kocho, mainly gram-positive, spore-forming bacteria of the genus Bacillus were found, among them also species of the Bacillus cereus group, Bacillus anthracis and Bacillus thurigiensis.