**8. Production considerations**

152 Fungicides for Plant and Animal Diseases

combinations and the combination method (co-culture or blend). Results of a two-way analysis of variance of disease with strain composition and combination method showed that significantly better dry rot suppression was obtained by co-cultures (30.3 + 2.4% relative disease) than by similar strain blends of pure cultures (41.3 + 2.4%) (P < 0.001). During a 3-year study, both biocontrol efficacy and consistency were assessed in 16 laboratory and small pilot trials simulating commercial storages. The 3-strain co-culture of *Pseudomonas fluorescens* strains S11:P:12, P22:Y:05, and S22:T:04 had a lower mean disease rating than the blend in 9 of 16 experiments examining control of the 3 diseases and sprouting. The co-culture led other treatments in incidences of significant malady reduction relative to the control: 14 of 16 attempts for co-culture, 11 of 16 attempts for blend, 10 of 13 attempts for pure S11:P:12, 8 of 13 attempts for S22:T:04, and 9 of 13 attempts for P22:Y:05. Using relative performance indices to rank treatment performance across all experiments, the co-culture treatment ranked significantly higher (69th percentile) than the blend (57th percentile). A synergy analysis suggested that co-culturing strains stimulated inter-strain activities to boost biocontrol efficacy and consistency, a feature not developed in strains grown separately and mixed just prior to addition to potatoes**.** Although the *E. cloacae* most often dominated co-cultures which included it, the other co-inoculated *P. fluorescens* populations persisted at significantly lower levels and apparently synergized the

There are a number of avenues by which the unique environment fostered by co-cultivation may improve biocontrol performance, and it is possible that inter-strain communication mechanisms are involved. Gram negative bacteria partners have been reported to regulate anti-microbial metabolite production via a signaling system referred to as "quorum sensing." Quorum sensing (QS) is mediated by population size and the accumulation of acylated homoserine lactones (AHL) which stimulate the bacterial populations to express genes responsible for metabolite production (Wood & Pierson, 1996). The local fermentation environment of the co-culture may synergize the impact of such signaling on subsequent biological control performance. Arrays of AHLs are known to be produced by many common rhizosphere bacteria, and they allow not only signaling between cells within a strain population, but also between cells of different strain populations (Pierson et al., 1998). An AHL-mediated QS system was noted to regulate cell surface properties, which was different from that noted for anti-microbial phenazine production (Zhang & Pierson, 2001). Soil bacteria have also been shown to degrade AHLs, such as via lactonase activity (Molina et al., 2003), a feature suggesting the potential for curative biocontrol of bacterial diseases. In addition to metabolite regulation and disease suppression, QS has been implicated in many other aspects of biocontrol activity, including: regulation of biofilm formation and rhizosphere colonization (Wei & Zhang, 2006); pathogen virulence, motility, and fitness (Licciardello et al., 2007); indoleacetic acid (IAA) plant growth hormone synthesis (Müller et al., 2009), perhaps pertinent to IAA accumulation by our *E. cloacae* strain S11:T:07; and

In addition to producing antifungal and sprout regulatory metabolites, we have recently identified the extracellular polysaccharide marginalan production by *P. fluorescens* S11:P:12 that not only improves its own survival during desiccation, but also that of co-inhabitants P22:Y:05 and S22:T:04 (Slininger et al., 2010a). This feature suggests the community benefit of one strain for others grown in association with it. In previous research by others, exopolysaccharides (EPS) have been associated with improved desiccation tolerance in

performance of the final population in suppressing dry rot disease.

induced systemic resistance (Pang et al., 2009).

For each strain of interest, the liquid culture production and biocontrol agent formulation processes must be designed to minimize cost and maximize production rate, yield, and quality, i.e. bioefficacy, storage stability, and host compatibility. The impact of liquid culture conditions (carbon and nitrogen sources, carbon-to-nitrogen ratio, nutrients, temperature, pH, dissolved oxygen), microbial physiology (growth state) and metabolites on the qualities of the biocontrol product will all need to be considered when designing the production processes for successful biocontrol products. To illustrate this, key findings of our research on this subject will be reviewed for a variety of our biological control agents under development, including but not exclusively our agents for post-harvest potatoes.

#### **8.1 Manipulation of growth, metabolism, and efficacy with culture conditions**

Prior research has shown that culture environment impacts metabolite accumulations and biocontrol agent quality. When strain *Pseudomonas fluorescens* 2-79 (NRRL B-15132) is efficiently delivered to the field in seed coatings, it colonizes the emerging root and produces the antibiotic, phenazine-1-carboxylic acid (PCA), as its primary means of suppressing take-all disease [incited by *Gaemannomyces graminis* var. *tritici* (Ggt)]. Our research has shown that metabolites (primarily PCA) present in liquid cultures of strain 2-79 cause significant germination losses (up to 64%) when included in seed coatings. In mass production of seed inocula, complete separation of cells from metabolites adds considerable expense and may not be feasible if metabolites are insoluble. For *Pseudomonas fluorescens* 2- 79, the phytotoxicity of the cell harvest can jeopardize the most economical method of application of the biocontrol agent, which is as a wheat seed coating. Our research showed that controlling fermentor environment allowed dramatic reduction of phytotoxic metabolite production. Fermentation conditions, such as dissolved oxygen, carbon source, pH, or temperature, were controlled to allow production of cells in a phytotoxin-free culture broth which could be used directly to treat seeds without sacrifice to either seed germination or to take-all disease control via PCA production in the rhizosphere (Slininger & Shea-Wilbur, 1995; Slininger et al., 1997a, 1998).

Biological Control Agents for Suppression

strain densities.

of Post-Harvest Diseases of Potatoes: Strategies on Discovery and Development 155

Though still synergistic to efficacy, *P. fluorescens* populations persisted at significantly lower levels than co-cultured *E. cloacae* S11:T:07 a facultative anaerobe with significant competitive advantage as dissolved oxygen is depleted. Growth suppression of multi-species bacterial populations in batch cultures by a single dominant strain has been referred to in the literature as the Jameson Effect and often involves production of specific inhibitors by one species against another. Future work may reveal inoculum population management and nutritional or physical environment management techniques that would allow testing the biocontrol efficacy of controllable population distributions of multi-genera co-cultures like *P. fluorescens* and *E. cloacae* strains, allowing investigation into the optimization of relative

**8.2 Influence of culture and physiological conditions on dry storage survival** 

the storage shelf-life of this biocontrol agent after air drying (Zhang et al., 2006).

Once the microbial biological control agent is harvested from the production culture, it will be necessary to formulate it. Formulations can be designed to meet a variety of objectives: to prevent biocontrol agent activity losses during storage; to facilitate convenient and efficacious delivery of the agent to the area where biocontrol is needed in the field; to promote insect biocontrol agent or plant-biocontrol agent compatibility; and to enhance the effectiveness of the biocontrol agent delivered. Examples are reviewed to illustrate formulation techniques devised by our research group to accomplish each of these goals for

**8.3 Formulating for storage stability and delivery** 

specific biocontrol problems.

The physiological state of cells upon harvest from the fermentation process was another factor that has been observed to influence the drying survival and shelf-life of strain 2-79 cells coated in 0.5% methylcellulose onto wheat seeds. Cells harvested from growth phase cultures (24-48 h) survived the drying process better than cells from stationary phase cultures (72-96 h), but stationary phase cells had a longer shelf-life than did growth phase cells. Our data suggest that the protective effect of residual sugar still present in growing cultures may explain why growth phase cells exhibited better survival of the drying process than did stationary cells that were deplete of sugar. The longer shelf-life of the stationary phase cells may come about via one or more of a variety of mechanisms that occur as a result of cell starvation and aging as reviewed by Slininger et al. (1996b, 1998). This feature has also been observed in our potato protective rhizobacteria and that influenced our choice of a 72-h harvest for our co-cultures in current research (Slininger et al., 2010a). The impact of cultivation conditions on biocontrol agent storage stability have been shown in virtually every other biocontrol system studied in our laboratory and not just those involving the control of plant diseases with rhizobacteria. Varying the carbon to nitrogen ratio and total carbon loading of a liquid medium for producing the bioherbicide *Colletotrichum truncatum* altered its dry storage stability as well as quantity, propagule type (conidia versus microsclerotia), and efficacy (Schisler et al., 1991; Jackson & Schisler, 1995). Yield and desiccation-tolerance of blastospores of the mycoinsecticide *Paecilomyces fumosoroseus* required appropriate concentrations of amino acids (Jackson, 1997; Jackson et al., 1997). Carbon-to-nitrogen ratio and carbon loading were found to influence the freeze-drying survival and Fusarium head blight suppressiveness of *Cryptococcus nodaensis* (now *C. flavescens*) OH 182.9 (Zhang et al., 2005); and cold shock during liquid cultivation increased

Culture environment also impacts the metabolism and efficacy of our potato protective rhizobacteria which have been reported to produce at least one antifungal component per strain (Burkhead et al., 1995). Our process for ranking dry rot antagonists relative to commercial potential involved growing the strains on three different liquid media of varying nutritional richness and then applying them to potato wounds challenged by the pathogen. The rank of candidate strains based on the liquid culture growth and also disease suppressiveness of the product was found to vary widely with the nutritional environment provided during production of the biological control agent. The metabolite profile of *E. cloacae* strain S11:T:07 has been studied in detail, and it is known to produce indoleacetic acid, phenyl acetic acid, and tyrosol. Concentrations of these metabolites influence both disease suppression and sprouting and vary in cultures with the nutritional environment (Burkhead et al., 1998; Slininger et al., 2004). As discussed in section 7 above, the cocultivation of multiple potato malady-suppressive strains gives rise to a unique fermentation environment that can yield a biocontrol product with improved efficacy and consistency (Slininger et al., 2010b). However, additional process optimization challenges may arise in accommodating population yields, storage stability, and efficacy. Although the performance benefits of co-cultivations to biocontrol performance had not been documented prior to our 2010 report, mixed Pseudomonad cultivations (with other *Pseudomonas* sp. or other genera) have been documented for many other applications (for example, Rodriguez & Gallardo, 1993; Kimura & Ito, 2001; Ashby et al., 2005; Kumar et al., 2006). More recently, Wu et al. (2009) examined the synergistic growth of a salt tolerant *Pseudomonas fluorescens* Rs-198 with another bacterium Rs-5 in co-culture that may have potential for application in fertilizer preparation. Co-cultures of potato-protective *P. fluorescens* strains could easily be designed to develop similar population densities even by simple adjustments in initial population densities to compensate for growth differences in strains as evaluated by a non-antibiotic selective plating technique described in Slininger et al. (2010b) (Figure 3).

Fig. 3. Growth of P. fluorescens strains S11:P:12, P22:Y:05, and S22:T:04 in duplicate coculture fermentor runs (Slininger et al., 2010b).

Culture environment also impacts the metabolism and efficacy of our potato protective rhizobacteria which have been reported to produce at least one antifungal component per strain (Burkhead et al., 1995). Our process for ranking dry rot antagonists relative to commercial potential involved growing the strains on three different liquid media of varying nutritional richness and then applying them to potato wounds challenged by the pathogen. The rank of candidate strains based on the liquid culture growth and also disease suppressiveness of the product was found to vary widely with the nutritional environment provided during production of the biological control agent. The metabolite profile of *E. cloacae* strain S11:T:07 has been studied in detail, and it is known to produce indoleacetic acid, phenyl acetic acid, and tyrosol. Concentrations of these metabolites influence both disease suppression and sprouting and vary in cultures with the nutritional environment (Burkhead et al., 1998; Slininger et al., 2004). As discussed in section 7 above, the cocultivation of multiple potato malady-suppressive strains gives rise to a unique fermentation environment that can yield a biocontrol product with improved efficacy and consistency (Slininger et al., 2010b). However, additional process optimization challenges may arise in accommodating population yields, storage stability, and efficacy. Although the performance benefits of co-cultivations to biocontrol performance had not been documented prior to our 2010 report, mixed Pseudomonad cultivations (with other *Pseudomonas* sp. or other genera) have been documented for many other applications (for example, Rodriguez & Gallardo, 1993; Kimura & Ito, 2001; Ashby et al., 2005; Kumar et al., 2006). More recently, Wu et al. (2009) examined the synergistic growth of a salt tolerant *Pseudomonas fluorescens* Rs-198 with another bacterium Rs-5 in co-culture that may have potential for application in fertilizer preparation. Co-cultures of potato-protective *P. fluorescens* strains could easily be designed to develop similar population densities even by simple adjustments in initial population densities to compensate for growth differences in strains as evaluated by a non-antibiotic selective plating technique described in Slininger et al. (2010b) (Figure 3).

Fig. 3. Growth of P. fluorescens strains S11:P:12, P22:Y:05, and S22:T:04 in duplicate co-

culture fermentor runs (Slininger et al., 2010b).

Though still synergistic to efficacy, *P. fluorescens* populations persisted at significantly lower levels than co-cultured *E. cloacae* S11:T:07 a facultative anaerobe with significant competitive advantage as dissolved oxygen is depleted. Growth suppression of multi-species bacterial populations in batch cultures by a single dominant strain has been referred to in the literature as the Jameson Effect and often involves production of specific inhibitors by one species against another. Future work may reveal inoculum population management and nutritional or physical environment management techniques that would allow testing the biocontrol efficacy of controllable population distributions of multi-genera co-cultures like *P. fluorescens* and *E. cloacae* strains, allowing investigation into the optimization of relative strain densities.

### **8.2 Influence of culture and physiological conditions on dry storage survival**

The physiological state of cells upon harvest from the fermentation process was another factor that has been observed to influence the drying survival and shelf-life of strain 2-79 cells coated in 0.5% methylcellulose onto wheat seeds. Cells harvested from growth phase cultures (24-48 h) survived the drying process better than cells from stationary phase cultures (72-96 h), but stationary phase cells had a longer shelf-life than did growth phase cells. Our data suggest that the protective effect of residual sugar still present in growing cultures may explain why growth phase cells exhibited better survival of the drying process than did stationary cells that were deplete of sugar. The longer shelf-life of the stationary phase cells may come about via one or more of a variety of mechanisms that occur as a result of cell starvation and aging as reviewed by Slininger et al. (1996b, 1998). This feature has also been observed in our potato protective rhizobacteria and that influenced our choice of a 72-h harvest for our co-cultures in current research (Slininger et al., 2010a). The impact of cultivation conditions on biocontrol agent storage stability have been shown in virtually every other biocontrol system studied in our laboratory and not just those involving the control of plant diseases with rhizobacteria. Varying the carbon to nitrogen ratio and total carbon loading of a liquid medium for producing the bioherbicide *Colletotrichum truncatum* altered its dry storage stability as well as quantity, propagule type (conidia versus microsclerotia), and efficacy (Schisler et al., 1991; Jackson & Schisler, 1995). Yield and desiccation-tolerance of blastospores of the mycoinsecticide *Paecilomyces fumosoroseus* required appropriate concentrations of amino acids (Jackson, 1997; Jackson et al., 1997). Carbon-to-nitrogen ratio and carbon loading were found to influence the freeze-drying survival and Fusarium head blight suppressiveness of *Cryptococcus nodaensis* (now *C. flavescens*) OH 182.9 (Zhang et al., 2005); and cold shock during liquid cultivation increased the storage shelf-life of this biocontrol agent after air drying (Zhang et al., 2006).

#### **8.3 Formulating for storage stability and delivery**

Once the microbial biological control agent is harvested from the production culture, it will be necessary to formulate it. Formulations can be designed to meet a variety of objectives: to prevent biocontrol agent activity losses during storage; to facilitate convenient and efficacious delivery of the agent to the area where biocontrol is needed in the field; to promote insect biocontrol agent or plant-biocontrol agent compatibility; and to enhance the effectiveness of the biocontrol agent delivered. Examples are reviewed to illustrate formulation techniques devised by our research group to accomplish each of these goals for specific biocontrol problems.

Biological Control Agents for Suppression

Storage Relative Humidity (%)

Formulation Sugar

Storage Time (d)

(P<0.05).

of Post-Harvest Diseases of Potatoes: Strategies on Discovery and Development 157

51 5.6 A 4.4 x 108 A 58.6 A 86 8.7 B 4.3 x 108 A 49.7 A

Lactose + BSA 5.8 A 3.9 x 106 A 56.4 A Lactose 7.0 AB 5.8 x 106 A 66.9 A Sucrose 7.2 AB 3.4 x 106 A 57.4 A Fructose 8.6 B 4.4 x 106 A 35.9

7 3.8 A 1.1 x 107 A 52.0 A 48 4.4 AB 3.5 x 106 B 47.2 A 83 7.1 B 2.4 x 106 B 55.5 A 145 13.4 C 1.3 x 106 B 62.3 A

aWithin each column and storage factor, means having letters in common are not significantly different

Table 2. Impact of storage conditions on drying survival of *Enterobacter cloacae* S11:T:07 in

**9. Filling the gaps to commercialization — A high-throughput screening** 

In conclusion, much progress has been made with methods of screening and selecting commercially useful biological control agents and in identifying key aspects of cultivation and formulation that impact biocontrol agent yield and quality. However, the development and optimization of integrated technologies to produce and deliver effective biological control agents remains a barrier to commercialization for many biocontrol agents. We have noted a myriad of variables associated with biocontrol agent cultivation, formulation, drying, storage, and reconstitution processes complicate agent quantity and quality maximization. To approach this problem, an efficient assay was conceived and applied using a 96-well microplate format to allow an integrated approach to optimizing these process variables. The basic high-throughput screening assay is depicted in Figure 4 and involves growing the BCA of interest (in flasks, fermentors, or microplates), formulating cells harvested from growth cultures, delivering microliter droplets of formulated cells to microplate wells (for rapid or slow drying), air- or freeze-drying droplets in the wells, storing plates, reconstituting dried cells, and then monitoring cell activity in terms of the rate of cell growth to a specified yield using a plate-reading spectrophotometer (Slininger & Schisler, 2003). Relevant variables (ingredients, temperature, etc.) are treated at each step of the assay process to view their individual and combined impact on resultant microbial activity,

the droplet drying screena (Slininger and Schisler, 2003)

**concept for optimizing biocontrol performance** 

Surviving Viable Cells (cfu/well)a

Relative Dry Rot Disease Rating (%)a

Time to A620=0.05 (h)a

The loss of microbial viability during storage is one of the most challenging barriers to overcome on the road to commercial success, especially for strains that do not form a resilient spore. Formulation matrices and storage temperature can play an important role in improving storage survival. For example, in the case of *Paecilomyces fumosoroseus* blastospores, calcined kaolin clay allowed significantly better drying survival and storage stability than other matrices tested (diatomaceous earth, talcs, corn starch, rice flour, and Mexican lime). Greater than 70% survival was retained after air drying and storage 42 days at 4°C, and near 20% after 21 days at 28°C was retained using 5% clay (Sandoval-Coronado et al., 2001). The trend of longer term storage survival with decreasing storage temperature has also held true for liquid formulations of *Pseudomonas fluorescens* and *Enterobacter cloacae*, our gram-negative bacterial biocontrol agents of potato dry rot and sprouting; cells frozen at -20°C in neutral buffer exhibited half-lives of 72-161 days, while those refrigerated at 4°C had half-lives of only 12-33 days (Slininger et al., 1997b). Another general finding of our work has been that the inclusion of culture broth with cells in stored formulations is often, but not always, detrimental to long term cell survival. For example, our gram-negative biocontrol agents for potatoes exhibited half-lives of 26-97 days when frozen in their culture broth and half-lives of 12-42 days when refrigerated in their culture broth. Thus, when frozen, the cells formulated in culture broth had poorer survival than cells in buffer, but when refrigerated, cell survival was similar in culture broth and buffer. When *Pseudomonas fluorescens* 2-79 was stored refrigerated in dried methylcellulose coatings of wheat seeds, the presence of the culture broth was again observed to be detrimental to drying and long-term cell survival, but the data showed that the presence of glucose in methylcellulose coatings with culture broth reduced cell losses upon drying (Slininger et al., 1996b). These examples illustrate the impact of formulation and storage conditions on biocontrol agent preservation and suggest both the aptitude and need for technology advancement in this area.

#### **8.4 Exopolysaccharide production and use as an** *in situ* **formulant**

Suppressive to potato diseases and sprouting, *Pseudomonas fluorescens* S11:P:12 (NRRL B-21133) produces a polysaccharide during liquid cultivation which was isolated, purified, and identified as marginalan (Slininger et al., 2010a). Dry storage results indicated that the presence of marginalan significantly reduced cell death after drying, such that the final stable viable cell density was 2.5 to 5 orders of magnitude greater, respectively, than if no marginalan were included with cells. Marginalan had no significant impact on disease or sprout suppression by strain S11:P:12, and its main benefit to biocontrol was viable cell preservation during drying and storage. When marginalan was formulated with other selected *P. fluorescens* strains P22:Y:05 and S22:T:04, as may occur in co-cultures, its benefits to drying and storage survival were again evident, though more subtle than observed for strain S11:P:12—perhaps because it was the most sensitive of the three to drying. Due to marginalan production, higher viscosity and higher fermentation power consumption for aeration and mixing will be needed to maximize viable cell yield in cultures containing S11:P:12. On the other hand, the polysaccharide offers to return value in terms of enhanced biological control as a cell desiccation protectant and should be considered in culture optimization schemes and for use in downstream formulation methodologies.


The loss of microbial viability during storage is one of the most challenging barriers to overcome on the road to commercial success, especially for strains that do not form a resilient spore. Formulation matrices and storage temperature can play an important role in improving storage survival. For example, in the case of *Paecilomyces fumosoroseus* blastospores, calcined kaolin clay allowed significantly better drying survival and storage stability than other matrices tested (diatomaceous earth, talcs, corn starch, rice flour, and Mexican lime). Greater than 70% survival was retained after air drying and storage 42 days at 4°C, and near 20% after 21 days at 28°C was retained using 5% clay (Sandoval-Coronado et al., 2001). The trend of longer term storage survival with decreasing storage temperature has also held true for liquid formulations of *Pseudomonas fluorescens* and *Enterobacter cloacae*, our gram-negative bacterial biocontrol agents of potato dry rot and sprouting; cells frozen at -20°C in neutral buffer exhibited half-lives of 72-161 days, while those refrigerated at 4°C had half-lives of only 12-33 days (Slininger et al., 1997b). Another general finding of our work has been that the inclusion of culture broth with cells in stored formulations is often, but not always, detrimental to long term cell survival. For example, our gram-negative biocontrol agents for potatoes exhibited half-lives of 26-97 days when frozen in their culture broth and half-lives of 12-42 days when refrigerated in their culture broth. Thus, when frozen, the cells formulated in culture broth had poorer survival than cells in buffer, but when refrigerated, cell survival was similar in culture broth and buffer. When *Pseudomonas fluorescens* 2-79 was stored refrigerated in dried methylcellulose coatings of wheat seeds, the presence of the culture broth was again observed to be detrimental to drying and long-term cell survival, but the data showed that the presence of glucose in methylcellulose coatings with culture broth reduced cell losses upon drying (Slininger et al., 1996b). These examples illustrate the impact of formulation and storage conditions on biocontrol agent preservation and suggest both the

aptitude and need for technology advancement in this area.

methodologies.

**8.4 Exopolysaccharide production and use as an** *in situ* **formulant** 

Suppressive to potato diseases and sprouting, *Pseudomonas fluorescens* S11:P:12 (NRRL B-21133) produces a polysaccharide during liquid cultivation which was isolated, purified, and identified as marginalan (Slininger et al., 2010a). Dry storage results indicated that the presence of marginalan significantly reduced cell death after drying, such that the final stable viable cell density was 2.5 to 5 orders of magnitude greater, respectively, than if no marginalan were included with cells. Marginalan had no significant impact on disease or sprout suppression by strain S11:P:12, and its main benefit to biocontrol was viable cell preservation during drying and storage. When marginalan was formulated with other selected *P. fluorescens* strains P22:Y:05 and S22:T:04, as may occur in co-cultures, its benefits to drying and storage survival were again evident, though more subtle than observed for strain S11:P:12—perhaps because it was the most sensitive of the three to drying. Due to marginalan production, higher viscosity and higher fermentation power consumption for aeration and mixing will be needed to maximize viable cell yield in cultures containing S11:P:12. On the other hand, the polysaccharide offers to return value in terms of enhanced biological control as a cell desiccation protectant and should be considered in culture optimization schemes and for use in downstream formulation


aWithin each column and storage factor, means having letters in common are not significantly different (P<0.05).

Table 2. Impact of storage conditions on drying survival of *Enterobacter cloacae* S11:T:07 in the droplet drying screena (Slininger and Schisler, 2003)

### **9. Filling the gaps to commercialization — A high-throughput screening concept for optimizing biocontrol performance**

In conclusion, much progress has been made with methods of screening and selecting commercially useful biological control agents and in identifying key aspects of cultivation and formulation that impact biocontrol agent yield and quality. However, the development and optimization of integrated technologies to produce and deliver effective biological control agents remains a barrier to commercialization for many biocontrol agents. We have noted a myriad of variables associated with biocontrol agent cultivation, formulation, drying, storage, and reconstitution processes complicate agent quantity and quality maximization. To approach this problem, an efficient assay was conceived and applied using a 96-well microplate format to allow an integrated approach to optimizing these process variables. The basic high-throughput screening assay is depicted in Figure 4 and involves growing the BCA of interest (in flasks, fermentors, or microplates), formulating cells harvested from growth cultures, delivering microliter droplets of formulated cells to microplate wells (for rapid or slow drying), air- or freeze-drying droplets in the wells, storing plates, reconstituting dried cells, and then monitoring cell activity in terms of the rate of cell growth to a specified yield using a plate-reading spectrophotometer (Slininger & Schisler, 2003). Relevant variables (ingredients, temperature, etc.) are treated at each step of the assay process to view their individual and combined impact on resultant microbial activity,

Biological Control Agents for Suppression

agent commercialization in the near future.

St. Joseph, MI: ASAE

*March 3-5,* Orlando, FL

30(5): 665-667

*Biomacromolecules* 6: 2106-2112

*Reviews of Phytopathology* 25: 67-85

*Biology and Biochemistry* 27(12): 1611-1616

**10. Disclaimer** 

**11. References** 

of Post-Harvest Diseases of Potatoes: Strategies on Discovery and Development 159

protective strain S11:T:07 are shown in Table 2 along with the results of other quality assays that can be carried out on microsamples, including viable cell counts and a wounded potato disease suppressiveness assay. We are currently applying such a flexible approach to allow further optimization of an integrated production process that would support biocontrol

The mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the U.S. Department of Agriculture. USDA is an equal opportunity provider and employer.

Abu-El Samen, F.M., Oberoi, K., Taylor, R.J., Secor, G.A. & Gudmestad, N.C. (2005).

Adiyaman, T., Schisler, D.A., Slininger, P.J., Sloan, J.M., Jackson, M.A. & Rooney, A.P.

ASAE (1990). Design and management of storages for bulk, fall-crop Irish potatoes.

Ashby, R.D., Solaiman, D.K.Y. & Foglia, T.A. (2005). Synthesis of short/medium-chain-

Baker, K.F. (1987). Evolving concepts of biological control of plant pathogens. *Annual* 

Behle, R.W., Tamez-Guerra, P., Shasha, B.S. & McGuire, M.R. (1999). Formulating

Burkhead, K.D., Schisler, D.A. & Slininger, P.J. (1995). Bioautography shows antibiotic

Burkhead, K.D., Slininger, P.J. & Schisler, D.A. (1998). Biological control bacterium

Cruz, L.P., Gaitan, A.L. & Gongora, C.E. (2006). Exploiting the genetic diversity of *Beauveria* 

use of strain mixtures. *Applied Microbiology and Biotechnology* 71: 916-926 Desjardins, A.E., Christ-Harned, E.A., McCormick, S.P. & Secor, G.A. ( 1993). Population

*pulicaris* from potato tubers. *Phytopathology* 83: 164-170

Boyd, A.E.W. (1972). Potato storage diseases. *Reviews of Plant Pathology* 51: 297-321

*American Journal of Potato Research* 82: 105-115

kinetics index rankings. *Plant Disease* 95(1): 24-30

Inheritance of mefenoxam resistance in selfed populations of the homothallic oomycete *Phytophthora erythroseptica* (Pethybr.), cause of pink rot of potato.

(2011). Selection of biocontrol agents of pink rot based on efficacy and growth

length poly(hydroxyalkanoate) blends by mixed culture fermentation of glycerol.

bioinsecticides to improve residual activity. *Proceedings of the Formulations Forum,* 

production by soil bacterial isolates antagonistic to fungal dry rot of potatoes. *Soil* 

*Enterobacter cloacae* S11:T:07 (NRRL B-21050) produces the antifungal compound phenylacetic acid in Sabouraud maltose broth culture. *Soil Biology and Biochemistry*

*bassiana* for improving the biological control of the coffee berry borer through the

structure and genetic analysis of field resistance to thiabendazole in *Gibberella* 

such as the speed of reaching logarithmic growth and a certain cell yield, such as in this research an optical density of 0.05 at a defined wavelength (620 nm). A kinetic activity assessment such as this is convenient to accomplish for the numerous samples that can potentially arise in multivariate experiments and is a good initial screen of biocontrol agent activity as it relates to viable cell concentration in combination with cell activity level (Figure 4).

Fig. 4. Droplet drying method to screen and optimize integrated biocontrol agent production process variables (Slininger and Schisler, 2003)

Numerous variables (culture and formulation ingredients, temperature, humidity, etc.) can be tested at each step of the assay process to view their individual and combined impact on the resultant microbial activity. The results of such an assay applied to our potato dry rot protective strain S11:T:07 are shown in Table 2 along with the results of other quality assays that can be carried out on microsamples, including viable cell counts and a wounded potato disease suppressiveness assay. We are currently applying such a flexible approach to allow further optimization of an integrated production process that would support biocontrol agent commercialization in the near future.
