Browsing by Author "Kifle, Medhin Hadish."

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Development of free-living diazotrophic (FLD) inoculants and their effects on crop growth.
(2008) Kifle, Medhin Hadish.; Laing, Mark Delmege.
In this study several free-living diazotrophs (FLD) were isolated and screened for their nitrogen fixing ability on a range of crops grown in greenhouse, hydroponics and field trials. Rhizosphere isolates of free-living diazotrophs (FLD) may be effective biofertilizer inoculants, and may improve plant health where crops are grown with little or no fertilizer, as is the case in the Developing World. FLD isolates from rhizospheric soils in KwaZulu-Natal were assessed by growing them on N-free media, which is a key isolation method. They were then evaluated for their nitrogenase activity by quantifying ethylene production from acetylene by gas chromatography (GC). The free living isolates that produced greater quantities of ethylene were detected by an acetylene reduction assay (ARA). These were further assessed for colony formation on N-free media with different carbon sources, and at a range of temperatures (20, 25 and 300C) and pH values (6.0, 7.0 and 8.0). Isolates G3 and L1 were identified using DNA sequencing by Inqaba Biotechnical Industries (Pty) Ltd as Burkholderia ambifaria Coenye et al, and Bacillus cereus Frankland, respectively. These isolates grew significantly better on an ethanol medium, at temperatures of 20, 25 and 300C and pHs of 6.0, 7.0 and 8.0. Isolates B3 (Burkholderia sp.) and D6 (Bacillus cereus Frankland) also grew well on an ethanol medium, but only at 200C and at a pH of 6.0 and 7.0, respectively, while Isolate E9 (Burkholderia cepacia Frankland) grew well on an ethanol medium only at 300C, and pH 6.0 and 7.0. Temperature and pH strongly influence FLD growth on N-free media using different carbon sources. Further trials were conducted to screen the best isolates under greenhouse condition, using both seed treatments and drenching application techniques onto several crops. The drenching application resulted in an increase in the growth and N-total of all the evaluated crops, relative to an unfertilized control. Growth and N-total of maize and sorghum increased with seed treatments, but did not increase the growth of lettuce and zucchini. Drenching of FLD isolates at 106cfu ml-1, applied on weekly basis, resulted in an increase in the growth of lettuce. Increased doses and frequency of application of the FLD bacteria resulted in a decrease in lettuce growth. This led to the conclusion that application of FLD bacteria at high doses and short intervals may create a situation where the applied FLD bacteria and the resident rhizosphere microbes compete for root exudates. High doses at low frequencies and low doses at high frequencies may be more effective on lettuce. Inoculation of Isolate L1 (B. cereus) at 106cfu ml-1 or in combination with Eco-T® (Trichoderma harzianum Rifai), significantly increased growth of lettuce. This result may have been due to nitrogen fixation, or to secretion of growth promoting substances by both the FLD and T. harzianum, and to biocontrol effects of Eco-T®. Application of Isolate L1 (B. cereus) at 106cfu ml-1 with or without Eco-T® was an effective tool for enhancing plant growth and nitrogen fixation. An FLD, Isolate L1 (B. cereus), was applied to lettuce plants together with a complete hydroponics fertilizer at 25% strength (Ocean Agriculture 3:1:3 (38) Complete), with the N level at 25mg l-1. These plants grew significantly better than the control plants grown on 25% of normal NPK fertilization, or with an inoculation of L1 alone. This indicates that it may be possible to integrate FLD applications with the application of low levels of commercial fertilizers, which is what resource poor farmers can afford.
Evaluation of diazotrophic bacteria as biofertilizers.
(2013) Kifle, Medhin Hadish.; Laing, Mark Delmege.;
Inoculation with diazotrophic bacteria is well documented as a means to enhance growth and increase yields of various crops, especially when used as an alternative or a supplement to the use of nitrogenous fertilizers and agrochemicals for sustainable agriculture. Nitrogen is the most limiting nutrient for increasing crop productivity, and the use of chemical sources of N fertilizers is expensive, and may contribute to environmental pollution. Therefore, there is a need to identify diazotrophic inoculants as an alternative or supplement to N-fertilizers for sustainable agriculture. The search for effective diazotrophic bacterial strains for formulation as biofertilizers has been going on for over 40 years and a number of inoculant biofertilizers have been developed and are commercially available. In the current study, 195 free-living diazotrophic bacteria were isolated from soils collected from the rhizosphere and leaves of different crops in different areas within the KwaZulu-Natal Province, Republic of South Africa. Ninety five of the isolates were selected for further screening because they were able to grow on N-free media using different carbon sources. Isolates that were very slow to grow on N-free media were discarded. Of these, 95 isolates were screened in vitro for growth promotion traits tests including tests for ammonia production and acetylene reduction. The best 20 isolates that were also able to reduce acetylene into ethylene were selected for growth-promotion trials on maize under greenhouse conditions. Of the 20 isolates, ten isolates enhanced (P = 0.001) growth of maize above the Un-inoculated Control. Molecular tests were conducted to identify the ten most promising isolates selected in the in vitro study. In the greenhouse study, these diazotrophic isolates were screened for their ability to enhance various growth parameters of maize (Zea mays L.), following various inoculation techniques (drenching, seed treatment, foliar spray and combination of these). Inoculations with the five best diazotrophic isolates by various methods of application increased dry weight and leaf chlorophyll content (P < 0.001, P = 0.001), respectively, compared to the Untreated Control. Although, all methods of application of diazotrophic inoculants used in this study resulted in increased dry weight and leaf chlorophyll content, combined methods of application (seed treatment + drenching) and sole application (seed treatment) were significantly more (P < 0.05) efficient. The best five most promising isolates were identified for growth promotion of maize under greenhouse conditions. They were also assessed for their effects on germination of wheat in vitro and were further tested in combination with various levels of nitrogenous fertilizer for growth-promotion of wheat (Triticum aestivum L.). These five isolates were also investigated for their potential to enhance growth and yields of maize and wheat crops in field trials, when combined with a low dose of nitrogenous fertilizer. These isolates were further studied for their contribution for enhancing plant growth through nitrogen fixation by predicting N content in leaves using a chlorophyll content meter (CCM-200) and correlated to extractable chlorophyll level at R2 = 0.96. In this study, relative to the Un-inoculated Control, the best five isolates enhanced growth of maize and wheat when combined with a 33% N-fertilizer levels for a number of growth parameters: increased chlorophyll levels and heights of maize, shoot dry weight of maize and wheat; and enhanced root and shoot development of these crops in both greenhouse and field conditions. The best contributions of diazotrophic bacteria was achieved by Isolate LB5 + 0% NPK (41%), V9 + 65% NPK (28.9%), Isolate L1 + 50% NPK (25%), Isolate L1 + 25%NPK (22%) and LB5 + 75% NPK (15%) undergreenhouse conditions. At 30 or 60 DAP, isolates with 33%N-fertilizer caused relatively higher dry weight than the 100%NPK. Inoculation of Isolate StB5 without 33N% fertilizer cuased significant (P<0.005) increases in stover dry weight. In field studies, inoculation of diazotrophic bacteria alone or with 33%N-fertilizer resulted in relatively greater increases of dry weight, stover dry weight, number of spikes and yield at different growth stages higher than the Un-inoculated or Unfertilized Control. However, the increases were not statistically significant. The use of microbial inoculants in combination with low doses of nitrogenous fertilizers can enhance crop production without compromising yields. The isolates obtained in this study can effectively fix nitrogen and enhance plant growth. The use of microbial inoculants can contribute to the integrated production of cereal crops with reduced nitrogenous fertilizer inputs, as a key component of sustainable agriculture.