Isolation and Characterization of Endophytes from Indegineous Tree Legumes
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Isolation and characterization of bacterial endophytes from indigenous tree legumes (Colophospermum mopane) and screening for their potential to produce plant growth hormones.
HIT 400 Project Proposal submitted in partial fulfillment of the
Requirements for the award of the degree of Bachelor of Technology (Hons) Degree in Biotechnology
To
Biotechnology Department
School of Industrial Sciences and Technology
Harare Institute of Technology (HIT)
By
Edinah Tembo`
H1210549T
Under the supervision of
Mrs P.D Chiunzi
1.0 Introduction
The status quo of Zimbabwe’s agriculture seeks to increase productivity along with a long term maintenance of a sustained yield. This aim however, can only be successfully achieved if there is a sufficient supply of plant growth hormones to the plants. Due to the market globalization, the exporting of the agricultural products is rapidly rising hence a need to meet the export demand. Zimbabwe has an abundant variety of indigenous tree legumes, unfortunately scientific taxonomic knowledge of the inhabiting endophytic species is limited and more has to be done to catch up with the rest of the world . The performance of the Zimbabwean economy has always been historically dependent and anchored on the production of the primary sector that is mining and agriculture. Although unpredictable rains continue to adversely affect the sector, the 2000 land reform programme made significant strides in employment creation and improving the livelihoods of the generality of Zimbabwean (Nyanga, 2013). Brachystegia spiciformis, commonly known as zebrawood, or Msasa, is a medium-sized African tree having compound leaves and racemes of small fragrant green flowers. The tree is broad and has a distinctive amber and wine red colour when the young leaves sprout during spring (August-September). It grows in savanna, both open woodland and closed woodland of Southern and Eastern Africa, mostly Tanzania, Zambia, Zimbabwe, Malawi and Mozambique. B. spiciformis has several medicinal applications including using the roots to treat dysentery and stomach problems . It is an important shade tree. The leaves are known to be a good fodder and would likely provide good mulch. B. spiciformis occurs in deciduous woodlands on ridges and escarpments (Orwa et al.2009). Fodder: Leaves are browsed by livestock.
Apiculture: Flowers provide a good source of pollen and nectar, giving an excellent honey, which granulates very slowly. Fuel: Trees are a good source of firewood and charcoal. Fibre: The inner bark is employed to make rope for roof ties, sacks, cloth, corn bins, beehives, and for other purposes. Timber: The wood is reddish-brown, coarse, not durable, difficult to season, subject to termite attack, tends to twist, split and warp. Even when treated, it is a rather inferior general purpose timber. It can be used for furniture and railway sleepers. Tannin or dyestuff: The bark is astringent, containing 13% tannin, and an extract of this is used by Africans as a finaldressing in tanning hides. It imparts a reddish colour to the finished product. Medicine: An infusion of the root provides treatment for dysentery and diarrhoea. A decoction is applied as an eyewash for conjunctivitis.
This study will benefit Zimbabwe’s agriculture by optimizing biological production of plant growth hormones if they are actually being produced by endophytes. Most indeginous tree legumes are found in the very dry regions, which cover over 60% of the country, however Msasa tree is found all over Zimbabwe. Hence introduction of commercial msasa planting would help boost this sector beyond the horizon as the specie’s growth is not solely dependent on rainfall and nature prescriptions .
Zimbabwean agriculture has the capacity if fully exploited of making significant contributions to the GDP and employment creation which are key antecedents to sustainable development. Approximately 20% of the economically active population are employed by the agricultural sector although much of it is employed only seasonal (Nyanga, 2013). The agricultural sector is second in terms of foreign currency generation, supplying up to 47% of exports. Improper or poor planning of the agricultural sector would impede national growth, employment creation, sustainable development and poverty would be widespread. All across the world people are in search of jobs yet jobs are very scarce especially in developing countries yet they are key to the wellbeing of families and provide people access to social protection for themselves and their families (Tripathi, 2004).
Endophytes are microorganisms (often fungi and bacteria) that live inside the tissues of living plants without causing any apparent harm to their host (Schulz et al, 1999). Endophytic bacteria are found in roots, stems, leaves, seeds, fruits, tubers, ovules and also inside legume nodules (Hallmann et al. 1997).
An understanding of the population dynamics of bacterial endophytes will enable more use to be made of their beneficial characteristics (Hallmann et al. 1997). This study was initiated however to isolate, characterize and correlate the presence of endophytes in different parts of the Msasa tree. They can be characterized using morphological, biochemical or molecular methods. Apparently there is no accessible information on isolation and characterization of bacterial endophytes associated with the Zimbabwean indigenous tree legumes, hence they are yet to be elucidated and be functionally characterized.
Endophytes have been recognized as a repository for novel secondary metabolites like ethylene and indole-3-acetic acid IAA of possible agricultural, pharmaceutical, and industrial importance. Thus, endophytes are a potential sources of new bioactive molecules and may be useful as agents for biotechnological processes (Schulz et al., 2002). Due to these features, endophytes are important from a bio prospection perspective, and with the use of biotechnological tools, it is plausible to develop products with economic potential from them (Strobel and Daisy, 2003; Suryanarayanan et al., 2009). In this study, we intend to explore the endophytic bacterial communities in the roots, leaves and twigs of C.mopane and their role in plant growth promoting activities along with antibiotic sensitivity, antibacterial and antifungal activities as well as the capability of inducing salt tolerance.
2.0 Problem statement
Zimbabwe has an abundant variety of indeginous tree legumes but has no comprehensive local endophytic classification database. According to Mpepereki and Pompi (2003) in Zimbabwe plant growth substances are chemically produced by companies like Agricura or Bayer and none have been biologically produced. The use of chemically derived rooting or shooting hormones such as indole 3-acetic acid, Giberillic acid, and auxins in potato farming is the most common practise but however these chemicals have long term detrimental effects on the environment. Moreso, many farmers cannot afford these synthetic plant growth hormones which are subject to fluctuating and ever-increasing prices (FAO, 2006). However if endophytes are characterized and are proven to produce phytohormones they can be used in the biological production of plant growth hormones or being incooperated as microorganisms in farming.
3.0 Justification
There are approximately …. Indeginous tree legumes species in Zimbabwe and they all are a potential habitat for endophytic bacteria. An understanding of population dynamics of bacterial endophytes will enable more use to be made of the endophyte’s beneficial characteristics. Investigations of the biodiversity of endophyte strains for novel metabolites may identify new products that can be used in agricultural and veterinary practices. The natural production of plant secondary metabolites for enhancing plant growth can reduce the excessive use of chemical-derived plant growth hormones which have long term negative effects to the ecosystem. Therefore characterization data generation of endophytic species found in Musasa tree is paramount in the quest of producing local species commercially. Knowledge of our natural indeginous tree legume endophytic database will help us unlock future research work in the biotechnology industry, hence creating employment.
4.0 Aim * To characterize endophytes in our indigenous tree legumes (Mopani tree)
4.1 Objectives
• To isolate the bacterial endophytes from the roots, stems and leaves of Mopani tree:
• To perform Morphological and biochemical characterization of the endophytes using staining techniques and biochemical tests.
• To determine if our indigenous tree legumes support the growth of endophytes and characterize the endophytes.
5.0 Literature review
Endophyte means “in the plant” (endon Gr. = within, phyton = plant). The usage of this term is as broad as its literal definition and spectrum of potential hosts and inhabitants, e.g. bacteria
(Kobayashi and Palumbo 2000), fungi (Stone et al. 2000), plants (Marler et al. 1999) and insects in plants (Feller, 1995), but also for algae within algae (Peters, 1991). Some bacteria and fungi enter the plants as endophytes and do not harm them by establishing symbiotic, mutualistic, commensalistic, and trophobiotic relationships (Nair and Padmavathy 2014).
Bacterial endophytes have been isolated from many different plants, mostly annual crop and pasture species. They have recently been reported from a range of host plants, including European deciduous and coniferous trees (Izumi et al. 2008), poplar (Ulrich et al. 2008), olives (Prieto and Mercado-Blanco 2008), and rice (Sun et al.2008). (Shenglian et al, 2011) performed the isolation and characterization of endophytic bacterium LRE07 from cadmium hyper accumulator Solanum nigrum L. and its potential for remediation. The endophytic bacteria are detected inside surface-sterilized plants or extracted from inside plants and have no visibly harmful effects on the plants (Hallmann et al. 1997). In most plants, roots have higher numbers of endophytes compared with above-ground tissues (Rosenblueth and Romero 2004).
Endophytic bacteria have been studied mainly after culturing in laboratory media, but a more complete scheme is emerging, using methods that do not require the bacteria to be cultured and that make use of the analysis of sequences from bacterial genes obtained from DNA isolated from inside plant tissues (Chelius and Triplett 2000). The functional characterization of the endophytes provides vital information about the contribution of the endophytes to the nutritional properties of the plants. However, the bacterial endophytes associated with the indigenous tree legumes are yet to be elucidated and functionally characterized.
Less is known about how a bacterium becomes a member of an endophyte community. Van Peer et al. (1990) found differences in several biochemical characteristics between external and internal Pseudomonas species and concluded that some form of selection by the plant or bacteria did occur. Preferences for the colonization of specific plant areas might be strain and species specific (Hallmann et al. 1997). Bacterial endophytes play an important role in plants' metabolic activities. They trigger various biochemical pathways in the host plant by producing physiologically important chemical compounds.
They produce a diverse range of secondary metabolites with vital medicinal values, and can be used in medicine, agriculture, and industry. (Strobel and Daisy 2003) Moreover, they actively participate in plant protection against environmental stresses and make them more adaptable (Nagarajkumar et al. 2004). Bacterial endophytes interact with plants through the production of phytohormones, siderophores, ammonia, HCN, phosphate solubilization (Rajkumar et al. 2006), and through lytic enzymes production (Nagarajkumar et al.2004).
6.0 Methodology
Endophytic bacterial isolates will be characterized on the basis of colony morphology and biochemical analysis. The morphological and biochemical characteristics of the isolates will be examined according to the Bergey’s Manual of Determinative Bacteriology (Holt et al. 1994). 1. Collection of plant material
Roots, leaves and twigs will be collected from Chitungwiza and they will be disinfected using 3 % Hydrogen peroxide, 70% ethanol and 1 % hypchlorite. After disinfection the sterilized roots, shoots and leaves will be sliced into small pieces and transferred to nutrient agar plates and incubated (25 °C) for 3-4 days. 2. Characterization of bacterial isolates (morphology and biochemical analysis)
The morphological and biochemical characteristics of the isolates will be examined according to the Bergey’s Manual of Determinative Bacteriology (Holt et al. 1994). Tests will include the Gram staining, Shape, Catalase, Oxidase, Glucose, Lactose, Maltose Mannitol, D-Mannose, Sucrose, Nitrate reduction, H2S production, Starch hydrolysis test. 3. PGP traits analysis: I. Testing for the production of IAA will be done upon culturing on Nutrient agar supplemented with LTryptophan and then centrifuged. The Supernatant will be mixed with orthophosphoric acid and Salkowski reagent and production of IAA will be confirmed by the development of a pink color (Brick et al. 1991). II. An antibiotic sensitivity test will be performed using antibiotic impregnated discs (6 mm dia.) containing streptomycin, tetracycline,chloramphenicol, amoxicillin, and neomycin using the Kirby Bauer disc-diffusion assay (Bauer et al. 1996 III. Antibacterial activity: All bacterial endophytes will be screened for their antibacterial properties following the cross-streak assay method (Williston et al. 1947) against Escherichia coli, Klebsiella pneumoniae, and Staphylococcus aureus. IV. HCN production: Bacterial isolates will be streaked on petri plates of solidified King’s B medium and the plates will then be sealed with parafilm and incubated (25±2 °C) for 72 h. The color change in the filter paper from will be visually assessed for production of HCN (Bakker and Schippers 1987).
7.0 Expected Output * Isolation of the bacterial endophytes from the roots, stems and leaves of Msasa tree * Characterization of the endophytes using morphological and biochemical tests . * Indigenous tree legumes should support the growth of endophytes. * Production of phytohormone indole acetic acid (IAA), inhibition bacterial growth production of HCN, and also solubilisation of phosphate.
9.0 Environmental Assessment
This project will have no negative impact on the environment in that it will not involve cutting down of the whole Mopani tree but only taking twigs, leaves and cutting a small portion of protruding roots. All the laboratory work will be under rigorous supervision.
10.0 Gantt Chat
| Oct | Nov | Dec | Jan | Feb | Mar | Apr | Literature review | | | | | | | | Protocol preparation | | | | | | | | Acquisation of the sample | | | | | | | | Laboratory work | | | | | | | | Data Analysis | | | | | | | | Characterization | | | | | | | | Document preparation. | | | | | | | |
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