Development of free-living diazotrophic (FLD) inoculants and their effects on crop growth.
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.