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Lack of technologies to produce and deliver effective biological control agents (BCAs) is a major barrier to their commercialisation. A myriad of variables associated with BCA cultivation, formulation, drying, storage and reconstitution processes complicate agent quality maximisation. An efficient assay using a 96-well microplate format to allow an integrated approach to optimising these process variables is presented. The assay involves growing the BCA of interest in flasks or fermentors, formulating cells harvested from growth cultures, delivering microlitre droplets of formulated cells to microplate wells, air-drying droplets, storing plates, reconstituting dried cells and monitoring the rate of cell growth to a specified yield using a plate-reading spectrophotometer. Spectrophotometer assessments of cell activity were significantly correlated with microdilution plate viable cell enumeration. Relevant variables (culture harvest age, cultivation and formulation ingredients, storage atmosphere and temperature) were tested with each step of the assay process to view their individual and combined impact on resultant microbial activity. The utility of this method to evaluate many treatments was demonstrated on seven strains ofPseudomonas fluorescensandEnterobacter cloacaeknown to suppress fungal diseases of wheat and potatoes. [ABSTRACT FROM AUTHOR]

A method to produce dried granules of Cryptococcus flavescens (formerly Cryptococcus nodaensis) OH 182.9 was developed and the granules evaluated for storage stability. Small spherical granules were produced and dried using a fluidized-bed dryer. A drying and survival curve was produced for the process of fluidized-bed drying at 30°C. The granules were dried to different moisture contents (4, 7, 9 and 12%) and evaluated for storage stability at 4°C for up to a year. These different moisture contents granules had the following respective water activities (0.22, 0.38, 0.47 and 0.57 a w). The results show the storage stability varied significantly across this moisture content range. The 9% moisture content sample had the best short-term stability (up to 4 months), while 4% moisture content had the best long-term survival (1 year). A desorption isotherm of C. flavescens was determined and modeled. The results of the storage stability and drying studies are interpreted in context of the desorption isotherm. [ABSTRACT FROM AUTHOR]

Introduction of US-8 genotypes of Phytophthora infestans has coincided with an increase in severity of potato late blight in North America. As alternatives to chemical fungicides, 18 bacterial strains patented as biological control agents (BCA) of both sprouting and Fusarium dry rot were cultivated in three liquid media and screened in wounded potato bioassays for their ability to suppress late blight incited by P. infestans (US-8, mating type A2). Washed or unwashed stationary-phase bacteria were mixed with fungal zoospores to inoculate potato wounds with 5µL containing ∼108 bacterial CFU/mL and 2×104 zoospore count/mL. Disease suppressiveness was evaluated after tubers were stored a week at 15°C, 90% relative humidity. One-fifth of the 108 BCA treatments screened, reduced late blight by 25-60%, including among other strains Pseudomonas fluorescens S22:T:04 (showing most consistency), P22:Y:05, S11:P:12 and Enterobacter cloacae S11:T:07. Small-scale pilot testing of these four strains, alone and in combination, was conducted under conditions simulating a commercial application. Suspensions of 4×104 P. infestans sporangia/mL were sprayed at a rate of 1.6 mL followed by 0.8 mL of bacteria treatment at ∼5×109 CFU/mL per each of 90 unwounded potatoes. Three replicate boxes per treatment (30 tubers per box) were randomized in storage and maintained 4 weeks at 7.2°C, 95% relative humidity. All BCA treatments significantly reduced disease; and unwashed bacteria outperformed those washed free of culture broth. Disease suppression ranged from 35% up to 86% the first test year and from 35 to 91% the second year. Highest overall performance rankings significantly above the control were achieved by the following strains in culture broth: four-strain mix > P. fluorescens S22:T:04> P. fluorescens S11:P:12. Combined with previous demonstrations of dry rot and sprout suppression, the consistent late blight control by these strains and strain mixtures suggests the commercial feasibility of a single treatment for broad spectrum suppression of post-harvest potato diseases and sprouting. [ABSTRACT FROM AUTHOR]

Fermentation, formulation and drying studies are necessary and important in order to simplify production, transportation, storage and application of biocontrol agents. Air-drying is a convenient and economical drying method for developing microbial biocontrol products. Experiments were designed to determine the effect of temperature shock during liquid cultivation on cell survival of a Fusarium head blight biocontrol agent Cryptococcus nodaensis OH 182.9 after air-drying. OH 182.9 cultures were grown at various temperatures in semi-defined complete liquid media, with cultures grown at 25°C for 48 h serving as the standard control culture condition. Harvested cultures were mixed with 10% diatomaceous earth (DE), vacuum filtered, air dried for 20 h at 60–70% RH, and stored at 4°C. In general, cells grown at 25°C for 20 h followed by cultivation at 15°C for 28 h survived air-drying better than control cells. The survival of cells subjected to heat shock at 31°C generally did not differ from control cells regardless of whether heat shock was applied at the late exponential or early stationary stage of growth. In another experiment designed to optimize the effect of cold temperatures during cultivation on subsequent survival of air-dried cells in DE at 4°C and room temperature (25°C), prolonged (28 h) cold shock at 10 and 15°C after incubation at 25°C for 20 h enhanced the storage stability (shelf-life) of a DE-formulated OH 182.9 product. In greenhouse tests, air-dried cells of OH 182.9 stored for 6 weeks at 4°C maintained a higher biocontrol efficacy than cells stored for 6 weeks at 25°C. [ABSTRACT FROM AUTHOR]

Fusarium head blight (FHB), caused by Gibberella zeae, is a devastating disease of wheat worldwide. Cryptococcus nodaensis OH 182.9 is an effective biocontrol agent for this disease. Development of a dried product of OH 182.9 would have potential advantages of ease of handling, favorable economics, and acceptance by end users. Isolate OH 182.9 was grown for 48 and 72 h in semi-defined complete liquid (SDCL) medium with carbon to nitrogen (C/N) ratios of 6.5:1, 9:1, 11:1, 15:1, and 30:1, and in SDCL C/N 30:1 media with varied carbon loadings of 7, 14, 21, and 28 g/liter. Total biomass production and cell survival at 15 days after freeze-drying were evaluated. Biomass production of OH 182.9 (CFU per milliliter) was not different for all cultivation time by medium C/N or carbon loading combinations. In general, cells harvested at 48 h survived freeze-drying better than those harvested at 72 h. Survival of freeze-dried cells was greatest for cells grown for 48 h in C/N 30:1 medium. Cells produced in C/N 6.5:1 medium generally exhibited the poorest survival. For the C/N 30:1 media, cells from 7 g/liter carbon loading medium harvested after 48 h had the best survival after freeze drying. The difference in freeze-dried cell populations between superior and inferior treatments was typically I to 2 log units at 15 days after freeze-drying. The biomass of OH 182.9 produced in SDCL with varied C/N ratios and in SDCL C/N 30:1 media with differing carbon loadings was tested for biocontrol efficacy against FHB in greenhouse studies. The biomass harvested from SDCL C/N 9:1, 11:1, and 15:1 media after 48 h significantly reduced symptoms of FHB. None of the treatments with cells harvested at 72 h consistently reduced FHB severity (P ≤ 0.05). Cells grown in SDCL C/N 30:1 media with 7 and 14 g/liter carbon loading significantly reduced FHB disease severity. Cells harvested from SDCL C/N 9:1, 11:1, and 30:1 with 14 g/liter carbon increased the 100-kernel weight compared with the disease control. The potential of improving OH 182.9 product quality via management of the nutritional environment of the production medium is demonstrated in this study. [ABSTRACT FROM AUTHOR]

Reports on the testing of bacteria strains that attack fungus on potatoes by the Agricultural Research Service.