The massive application of chemical fertilizers to support crop production has resulted in soil, water, and air pollution at a global scale. In the same time, this situation escalated consumers' concerns regarding quality and safety of food production which, due to increase of fertilizer prices, have provoked corresponding price increase of food products. It is widely accepted that the only solution is to boost exploitation of plant-beneficial microorganisms which in conditions of undisturbed soils play a key role in increasing the availability of minerals that otherwise are inaccessible to plants. This review paper is focused on the employment of microbial inoculants and their production and formulation. Special attention is given to biotechniques that are not fully exploited as tools for biofertilizer manufacturing such as microbial co-cultivation and co-immobilization. Another emerging area includes biotechnological production and combined usage of microorganisms/active natural compounds (biostimulants) such as plant extracts and exudates, compost extracts, and products like strigolactones, which improve not only plant growth and development but also plant-microbial interactions. The most important potential and novel strategies in this field are presented as well as the tendencies that will be developed in the near future.

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Unexploited potential of some biotechnological techniques for biofertilizer production and formulation

The cry gene content of Bacillus thuringiensis subsp. aizawai HD-133 was analyzed by a combination of high-pressure liquid chromatography (HPLC) and exclusive PCR. A total of six cry genes were detected in genomic DNA purified from HD-133, four from the cry1 family (cry1Aa, cry1Ab, cry1C, and cry1D) as well as a gene each from the cry2 (cry2B) and the cry1I families. To directly determine which genes were expressed and crystallized in the purified parasporal inclusions, solubilized and trypsinized HD-133 crystals were subjected to chromatographic separation by HPLC. Only three proteins, Cry1Ab, Cry1C, and Cry1D, were found, in a 60/37/3 ratio. Dot blot analysis of total mRNA purified from HD-133 showed that both the cry2B and cry1I genes, but not the cry1Aa gene, were transcribed. Cloning and sequencing of the cry1Aa gene revealed an inserted DNA sequence within the cry coding sequence, resulting in a disrupted reading frame. Taken together, our results show that combining crystal protein analysis with a genetic approach is a highly complementary and powerful way to assess the potential of B. thuringiensis isolates for new insecticidal genes and specificities. Furthermore, based on the number of cryptic genes found in HD-133, the total cry gene content of B. thuringiensis strains may be higher than previously thought.

A holistic approach for determining the entomopathogenic potential of Bacillus thuringiensis strains

Bacillus thuringiensis (Bt) has been used for control of lepidopteran, dipteran and coleopteran insects for over three decades. Novel Bt strains harbouring new types of insecticidal genes are being discovered worldwide. Recombinant strains with enhanced toxicity and broadened insecticidal spectrum have been constructed. To increase the field persistence of insecticidal crystal proteins (ICPs), alternative modes of their delivery in Pseudomonas sp. and endophytes have been developed. ICPs have been modified by site-directed mutagenesis to improve their insecticidal efficacy. Higher yields of ICPs have been achieved by use of strong expression promoters and other regulatory elements. Gene-disabling of the sporulation-specific protease has led to yield enhancement of ICPs. Interestingly, Bt toxins have been found to act synergistically with some other pesticidal agents. Optimization of fermentation conditions is an essential requirement for cost-effective commercial production of Bt biopesticides. The environmental impact of deployment of genetically engineered biopesticides has been assessed. Recombinant Bt strains that do not carry any non-Bt DNA, endophytes, encapsulation in killed bacteria (such as Pseudomonas) and asporogenous Bt strains are ecologically safe approaches. Efficient resistance management strategies require judicious use of Bt transgenic plants in conjunction with refugia and Bt biopesticides in an Integrated Pest Management (IPM) program.

Molecular approaches towards development of novel Bacillus thuringiensis biopesticides

Cost-effective production of Bacillus thuringiensis by solid-state fermentation

Bacillus thuringiensis (Bt) is a Gram-positive bacterium naturally found in soil, water and grain dust, and can be cultivated in liquid, solid and semi-solid media. The objective of this work was to test different media to grow B. thuringiensis. The seed culture (strain 344, B. thuringiensis tolworthi, belonging to Embrapa Maize and Sorghum Microorganism Bank) was produced using shake flasks and grown in LB medium plus salts during 18 hours, incubated on a rotary shaker at 200 revolutions per minute (rpm) at 30°C for 96 hours. Medium 1 was composed of: Luria Bertani (LB) plus salts (FeSO 4 , ZnSO 4 , MnSO 4 , MgSO 4 ), and 0.2% glucose; medium 2 was composed of 1.5% glucose, 0.5% soybean flour plus salts; and medium 3 was composed of liquid swine manure at 4% and 0.2% glucose. All three media were sterilized and inoculated with B. thuringiensis tolwothi (seed culture) at a stirrer speed of 200rpm, for 96 hours at 30°C. The pH was measured at regular intervals, viable spores were counted as c.f.u/mL, cell mass expressed in g/L- lyophilized, and spore counting per mL of medium. All three media showed pH variation during the fermentation process. Media 1 and 2 showed a tendency to shift toward a basic pH and medium 3 to an acidic pH. Media 1 and 2 showed the highest number of viable spores, 2.0 x 10 8 c.f.u/mL, within the 96 hours of incubation, however medium 2 showed a biomass dry weight of 1.18g/L. During the fermentation period, medium 1 showed the highest spore concentration, 1.4 x 10 9 spores/mL after 96h of fermentation. Efficiency against S. frugiperda first instar larvae showed that all Bt produced in all three media killed above 60% in the highest concentrations.

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O.N. Morris

Papers

Suitability of 30 Agricultural Products and By-Products as Nutrient Sources for Laboratory Production of Bacillus thuringiensis subsp. aizawai (HD133)