Percy Chimwamurombe

The morama bean (Tylosema esculentum): a potential crop for southern Africa.

The morama bean is an underutilized leguminous oilseed native to the Kalahari Desert and neighboring sandy regions of Botswana, Namibia, and South Africa (Limpopo, North-West, Gauteng, and Northern Cape provinces), and forms part of the diet of the indigenous population in these countries. It is also known as gemsbok bean, moramaboontjie, elandboontjie, braaiboonjie, marama, marumana, tsi, tsin, gami, and ombanui. It is reported as an excellent source of good quality protein (29-39%); its oil (24-48%) is rich in mono- and di-unsaturated fatty acids and contains no cholesterol. Morama is a good source of micronutrients such as calcium, iron, zinc, phosphate, magnesium, and B vitamins including folate. It is also reported to be a potential source of phytonutrients including phenolic compounds (e.g., tannins), trypsin inhibitors, phytates, and oligosaccharides, components which have been shown in other foods to contribute to health in particular, prevention of noncommunicable diseases such as cardiovascular diseases, diabetes, and some cancers. From a nutritional and health perspective, the morama bean has potential commercial value as a cash crop and value-added products, particularly in the communities where it is found.

Isolation and characterization of culturable seed-associated bacterial endophytes from gnotobiotically grown Marama bean seedlings

Marama bean (Tylosema esculentum) is an indigenous non-nodulating legume to the arid agro-ecological parts of Southern Africa. It is a staple food for the Khoisan and Bantu people from these areas. It is intriguing how it is able to synthesize the high-protein content in the seeds since its natural habitat is nitrogen deficient. The aim of the study was to determine the presence of seed transmittable bacterial endophytes that may have growth promoting effects, which may be particularly important for the harsh conditions. Marama bean seeds were surface sterilized and gnotobiotically grown to 2 weeks old seedlings. From surface-sterilized shoots and roots, 123 distinct bacterial isolates were cultured using three media, and identified by BOX-PCR fingerprinting and sequence analyses of the 16S rRNA and nifH genes. Phylogenetic analyses of 73 putative endophytes assigned them to bacterial species from 14 genera including Proteobacteria (Rhizobium, Massilia, Kosakonia, Pseudorhodoferax, Caulobacter, Pantoea, Sphingomonas, Burkholderia, Methylobacterium), Firmicutes (Bacillus), Actinobacteria (Curtobacterium, Microbacterium) and Bacteroidetes (Mucilaginibacter, Chitinophaga). Screening for plant growth-promoting activities revealed that the isolates showed production of IAA, ACC deaminase, siderophores, endoglucanase, protease, AHLs and capacities to solubilize phosphate and fix nitrogen. This is the first report that marama bean seeds may harbor endophytes that can be cultivated from seedlings; in this community of bacteria, physiological characteristics that are potentially plant growth promoting are widespread.

Isolation and Screening of Rhizosphere Bacteria from Grasses in East Kavango Region of Namibia for Plant Growth Promoting Characteristics

A diverse group of soil bacteria known as plant growth promoting rhizobacteria (PGPR) is able to inhabit the area close to plant roots and exert beneficial effects on plant growth. Beneficial interactions between rhizospheric bacteria and plants provide prospects for isolating culturable PGPR that can be used as bio-fertilizers for sustainable crop production in communities that cannot easily afford chemical fertilizers. This study was conducted with the aim of isolating rhizospheric bacteria from grasses along the Kavango River and screening the bacterial isolates for plant growth promoting characteristics. The bacteria were isolated from rhizospheres of Phragmites australis, Sporobolus sp., Vetiveria nigritana, Pennisetum glaucum and Sorghum bicolor. The isolates were screened for inorganic phosphate solubilization, siderophore production and indole-3-acetic acid (IAA) production. The nitrogen-fixing capability of the bacteria was determined by screening for the presence of the nifH gene. Up to 21 isolates were obtained from P. australis, Sporobolus sp., S. bicolor, P. glaucum and V. nigritana. The genera Bacillus, Enterobacter, Kocuria, Pseudomonas and Stenotrophomonas, identified via 16S rDNA were represented in the 13 PGPR strains isolated. The isolates exhibited more than one plant growth promoting trait and they were profiled as follows: three phosphate solubilizers, four siderophore producers, eight IAA producing isolates and five nitrogen-fixers. These bacteria can be used to develop bio-fertilizer inoculants for improved soil fertility management and sustainable production of local cereals.

Impacts of mine dump pollution on plant species diversity, composition and structure of a semiarid savanna in Namibia

Effects of mine dump pollution on semiarid savanna vegetation were investigated in Kombat, Namibia. Vegetation structure, species richness, composition and diversity were compared between polluted and control sites. Concentrations of arsenic, chromium, copper, lead and zinc in soils were significantly higher closer to a 99-year-old mine dump than in a control site and exceeded internationally recommended guidelines by as high as 11 times. Mine dump pollution has significantly reduced woody plant densities, species richness and diversity in the affected area due to disappearance of pollution-sensitive species and preponderance of tolerant ones. Species composition and structure of vegetation close to the mine dump significantly changed, possibly due to negative impacts of heavy metals on recruitment as pollution-sensitive species died off, whereas tolerant species invaded the vacated ecological niches. Ordination analyses confirmed a strong pollution gradient, with measured heavy metals accounting for 57.3% of the observed variation in species data, suggesting that other factors are also important determinants in the area. It is recommended that the mine dump and its surrounds should be rehabilitated and regular monitoring should be carried out to remedy the situation and arrest further pollution of the surrounding environment.

Domestication of [Tylosema esculentum (Burchell) Schreiber] (Marama bean): A work in Progress in Namibia

Marama bean [Tylosema esculentum (Burchell) Schreiber] is indigenous to the dry parts of Southern Africa. It is a staple food for the Khoisan and Bantu people from those areas. In Namibia it grows wild mainly in Omaheke and Otjizondjupa regions. Marama, which has not been domesticated, has a large tuber and pods containing 1-2 oil and protein-rich seeds with a nutritional value similar to soybean. Protein content is 3039% (similar to soy bean) and oil content is 30-43%. In addition it produces a edible tuber tht is rich in starch. Unfortunately, harvesting in the wild is very extensive and random by local people with such intensity that some genotypes of marama bean are becoming endangered with extinction or have already become extinct. This article reviews the current body literature on marama bean, domestication of selected major legumes and lastly reports on the progress made in the initial attempts to domesticate this plant by local communities in Namibia. The process starts off selecting marama genotypes with superior traits. These traits include early germination and number of seeds per pod. The University of Namibia has been developing marama for its introduction as a crop alternative for dry lands of Namibia and the Kalahari sandy regions of Southern Africa and also as a response to climate change effects where crops such as maize and millets have been failing due to alternating floods and drought. Marama has a huge potential to address the problem of malnutrition and hunger in the arid areas of Southern Africa, especially knowing that under climate change Africa will become drier and drier.

Effects of plant growth promoting bacterial isolates from Kavango on the vegetative growth of Sorghum bicolor.

Sorghum is an important cereal that is processed into a variety of foods and leisure beverages across the African continent. Low sorghum yields experienced in agriculture is a result of major production constraints such as soil nutrient deficiency and plant disease. It is important that the methods for crop production are of a sustainable nature as the chemical fertilizers in current use are detrimental to the natural environment. The aim of this study was to determine the effects of plant growth promoting (PGP) bacteria on growth of Sorghum bicolor. PGP bacteria isolated from the rhizosphere of Pennisetum glaucum (Pseudomonas stutzeri ACM2-32, Kosakonia cloacae FCM2-50, Bacillus subtilis ASM1-59 and Bacillus amyloliquefaciens LSM1-61) and S. bicolor (Stenotrophomonas maltophilia LCS2-11) plants in Kavango (Namibia), were used as peat-based inoculants to evaluate their effects on the growth of S. bicolor. The combination treatment T9 (B. amyloliquefaciens LSM1-61: K. cloacae FCM2-50: P. stutzeri ACM2-32) significantly (p = 0.032) enhanced the biomass of S. bicolor as compared to the water control. Single inoculants consisting of S. maltophilia LCS2-11, K. cloacae FCM2-50 and B. amyloliquefaciens LSM1-61 and combination inoculants T7, T8 and T9, enhanced S. bicolor root biomass as much as a commercial fertilizer control. These results indicate that the plant growth promoting bacteria induced a beneficial effect on growth of sorghum seedlings. The future work involves testing these promising inoculants on growth of these sorghum plants to maturity stage to determine effects on seed yield over three seasons in multi-location trials. Key words: Rhizosphere bacteria, peat based inoculants, plant growth promoting bacteria, Sorghum bicolor.

Isolation and Identification of Culturable Fungal Species Associated with Disease in Vachellia tortilis in Namibia

Vachellia tortilis is a tree belonging to the family Fabaceae that inhabits high temperature and dry environments of the world. It is ecologically and economically significant amongst others of the genera Vachellia and Senegalia. It is native to Namibia and several other countries. Various parts of the V. tortilis plant are used for different therapeutic purposes both in the traditional and pharmacological settings. However, V. tortilis is vulnerable to pathogenic infection to which they lack natural resistance and little is known regarding the pathogens responsible for such infections. The aim of the study was to isolate and identify the fungal species associated with twig dieback disease in V. tortilis. Pure cultures from diseased V. tortilis were grown using potato dextrose agar (PDA) and DNA was subsequently isolated and later amplified in a PCR reaction using ITS1 and ITS4 primers. Sequencing and BLAST search revealed the identity of the isolates as; Penicillium rubefaciens, Penicillium herbarum, Trichoderma longibrachiatum and Trichoderma harzianum. Results indicated that P. herbarum was associated with disease symptoms in A. tortilis, T. longibrachiatum and T. harzianum were antagonistic fungi while the effect of P. rubefaciens on V. tortilis remained unknown. It is recommended that further investigations using Koch’s postulates should be performed on V. tortilis using the isolates.

Isolation and Characterization of Fungi Associated with Disease Symptoms on Ziziphus mucronata Leaves and Phaseolus vulgaris Pods in Windhoek, Namibia

Detection of phytopathogens that are involved in causing disease symptoms in plants and crops is of prime importance as a key step in disease treatment and management. Ziziphus mucronata is a species endemic in temperate and tropical climates and used traditionally in the treatment of infectious diseases. The common bean (Phaseolus vulgaris) is a rich source of nutrients for the human diet. Just like most crops, it is not immune to fungal diseases and reports had been received of P. vulgaris showing signs on disease. The aim of this study was to isolate and characterize the fungal species associated with the disease symptoms in the Z. mucronata and P. vulgaris. Fungal species where isolated from surface-sterilised symptomatic bark of Z. muconata and fresh green bean pods. These where grown on petri dishes containing Potato Dextrose Agar and incubated at room temperature. Pure cultures where then obtained by transferring small segments of fungal growth to a new petri dish that contained PDA. During DNA extraction the pure cultures where first homogenized using liquid nitrogen and then the rest of the extraction carried out as stipulated in the Zymo extraction kit. The Nanodrop was used for quantifying the DNA and amplification of the conserved Internal Transcribed Spacer (ITS) region of Ribosomal RNA genes was carried out using ITS1 and ITS2. The PCR products were sequenced at Inqaba Biotech Industries in South Africa. The obtained sequences were then compared by alignment with known sequences in the Genbank using Basic Local Alignment Search Tool (BLAST). The BLAST searches were able to reveal the fungi isolated from the Z. mucronata as Fusarium penzigii and Fusarium dimerum while the fungi isolated from P. vulgaris shown to be Phoma destructiva, with 100 %, 95% and 100% sequence similarity respectively. The next step in this work is carry out Koch‘s postulates to determine which of this fungi is the causal agent of the observed diseases symptoms in order to start a targeted diseases management programme.

Molecular identification of fungal species associated with leaf lesions of marama bean seedlings in Namibia

During one of our greenhouse experiments on the marama bean plant (Tylosema esculentum) at the University of Namibia, various signs of necrosis and spotting were observed on leaf tissues of the newly developed seedlings. In this study, we hereby describe findings of the investigations undertaken to attempt and determine the possible causative agent(s) of such plant leaf infections. In brief, infected seedling leaves were first surface-sterilized and then used in form of leaf-discs (2 x 2 mm), to inoculate potato dextrose agar (PDA). Alongside this, control leaves were also similarly treated and overlaid onto PDA. While fungal growths were observed on all infected leaf-discs, no growth was observed on control discs. For each of the various fungal strains growing on the infected leaf discs, pure cultures were obtained by repeatedly sub-culturing the strains onto PDA. Subsequently, single spore cultures were also aseptically isolated from each of the obtained pure cultures and further developed into mycelia through inoculation and incubation in potato dextrose broth (PDB). All in all, a total of eight single spore cultures were obtained from the overall inoculations and sub-culturing. Subsequently, the total genomic DNAs of each of the obtained single spore cultures were isolated followed by amplification of their internal transcribed spacer (ITS) regions. The amplified ITS regions were then sequenced followed by a comparison of their nucleotide patterns with the GeneBank. Such a comparison then revealed the presence of a complex of several fungal isolates with eight commonly known species: Penicillium brevicompactum, Epicoccum sorghi, Rhizopus stolonifer, Alternari solani, Fusarium equiseti, Penicillium olsonii, Fusarium chlamydosporum and Fusarium incarnatum. These fungal species are commonly known to cause various diseases and infections in legumes and other agronomically important crops. Ideally, the presence of these fungal species in marama bean and their involvement in leaf tissue decay should be noted with great concern and interest since this plant has recently been proposed as a potential leguminous crop for possible adoption and utilization in domestication efforts. Key words: Marama bean, Tylosema esculentum, fungi, Namibia, leaf infection, internal transcribed spacer (ITS).

Co-infection of Tylosema esculentum (Marama bean) seed pods by Alternaria tenuissima and a Phoma spp.

Marama bean, Tylosema esculentum , is an endemic, perennial wild tuberous Fabaceae, widely distributed in the Southern Kalahari (Namibia, Botswana and to a lesser extends in South Africa). It is well adapted to the deep sandy soils of the Kalahari. It is drought avoiding. It has been valued by the indigenous people of the semi arid land of the Kalahari for its nutritional and medicinal qualities. The aim of the study was to determine the identity of fungal pathogens that occur on marama bean. Diseased pods presenting necrotic spots with dense sporulation in the centre were collected. They were subjected to fungal isolation using potato dextrose agar (PDA) at room temperature under fluorescent light for five days. Two fungi with different cultural conidial morphology were isolated. Single spores from the two cultures were separately inoculated on PDA to obtain a pure culture. Genomic fungal DNA was extracted from fresh mycelium using the cetyl trimethylammonium bromide (CTAB) method and was used as a template in internal transcribed spacer (ITS) PCR amplification. Based on the sequence analysis of the 5.8S ribosomal DNA and the ITS, the two isolates associated with necrotic Marama pods were identified as Alternaria tenuissima and Phoma spp. Isolate Po72 due to the high sequence homology, which was 99% in both cases. To our knowledge, this is the first report describing the presence of these two fungi on Marama bean. Key words : Marama, ITS, fungal pathogen, Tylosema esculentum .