1a.Objectives (from AD-416):
Develop a new energy efficient and simplified manufacturing process to mass-produce the aflatoxin biocontrol products as a low-cost formulation to increase affordability of the product by resource-poor farmers in the US and abroad.
1b.Approach (from AD-416):
1. Sorghum based formulations for atoxigenic biocontrol strains will be optimized in laboratory studies that will assess treatments (preconditioning steps) to the raw sorghum required for optimal spore yield.
2. Adaptation of formulating materials generally regarded as safe to the manufacture of biocontrol products will be explored with the objective of adapting diverse seed coating technologies to the low cost production of atoxigenic strain biological control products of use to farmers with low resources in Africa and the US.
3. Advanced Pyrosequencing techniques will be applied to determining optimal strain mixtures and resistance of formulations to contaminating strains from the environment and in so doing allow the exploration of cost effective multi-strain formulations.
4. Different sorghum types as the food base for atoxigenic strain formulations will be investigated along with potential adjuvants.
5. Problems associated with scaling up and shelf stability of products will be explored within the time frame of the proposed study.
Several studies were undertaken to develop a new energy efficient and simple manufacturing process to mass-produce the aflatoxin biocontrol product Aflasafe as a low-cost formulation to increase affordability of the product by both resource-poor farmers and U.S. farmers. The first atoxigenic strain commercialized for biological control of aflatoxin contamination is still produced by a farmer run and financed organization in the original manufacturing facility constructed of expensive custom designed equipment over a decade ago. During the current project, a manufacturing process was developed that utilizes inexpensive, off the shelf equipment used worldwide by the planting seed industry. In addition to low cost equipment, the process also has a greatly reduced energy footprint and does not require highly trained personnel. While developing the new process, a series of experiments were performed to obtain the desired information on large scale production of atoxigenic formulations. These included empirical tests to determine best conditions and practices for production of spores for formulations on sorghum grain in contrast to the wheat used in the U.S. The results are a process readily adapted to Africa and much less expensive than that in current use. Cost savings come primarily from use of sorghum instead of wheat, elimination of sterilization step, and increased speed of manufacture. Pretreatment of the sorghum was found to be necessary for production of formulations with good spore yield. Milo varieties varied widely in ability to support sporulation by the biocontrol fungi. Polymers and color coatings used routinely in the commercial seed industry worked well for coating killed sorghum with the biocontrol fungi. Formulations yielded excellent of spores. Natural polymers such as milk also worked well as did gum Arabic. Several adjuvants were also investigated including common salt (NaCl), several sugar products, and various milk products and gums. However, commercial materials currently used in the seed industry appear the easiest to adapt. To determine the influence of mixing atoxigenic strains into a single formulation on proportions of spores produced by each of the isolates on the final product, sorghum was coated with formulations containing spore mixtures. Pyrosequencing (a simple technique to analyze and quantitate DNA sequence) was used to quantify ratios of atoxigenics in the spores produced by each formulated product. Tests indicate that sorghum can be coated with appropriate ratios of four different atoxigenics in order to develop a product that will yield the desired ratios of spores in agricultural fields. This will avoid the need to make product with each atoxigenic separately and mix the four products after. The ability of the formulated AflaSafe product to exclude soil isolates of the fungus Aspergillus flavus was tested by incubating sorghum product on soil infested with a toxigenic isolate. Even when soil was infested with 5,000 CFU g-1 soil, the toxin producer comprised a small proportion of total conidia yielded. Note additional studies with higher concentrations of atoxigenics may provide insight into importance of conidia concentration on product quality. The program is monitored by periodic reports, presentations, and internet communication.