**7. Co-cultivation of strains — The next generation**

150 Fungicides for Plant and Animal Diseases

fungal zoospores to inoculate potato wounds. 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 (NRRL B-21053), S11:P:12 and *Enterobacter cloacae* S11:T:07, the later known to produce IAA. Small-scale pilot testing of these four strains, alone and in combination, was conducted under conditions simulating a commercial application. All four treatments significantly reduced disease; and unwashed bacteria outperformed those washed free of culture broth, indicating a role of metabolites such as IAA. 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 utility of a single treatment for broad spectrum suppression of post-harvest

Pink rot disease occurs in potato growing regions around the world and is caused primarily by the oomycete *Phytophthora erythroseptica* Pethybr. Losses of over 50% of the total harvest can result from tuber contamination by either pink rot or late blight (Secor & Gudmestad, 1999). All underground portions of potato plants can be infected. Root and stem infections can result in plant wilting and death. Though some evidence indicates that there is limited genetic diversity in North American isolates of *P. erythroseptica* (Peters et al., 2005), infections initiated after tuber harvest are difficult to control. Most commercially grown potato cultivars in Canada and the United States are susceptible to pink rot and breeding efforts against this disease have been minimal (Peters et al., 2004). Mefenoxam, a phenylamide fungicide that formerly was effective in reducing the disease in storage, has lost much of its effectiveness (Taylor et al., 2006) due to widespread genetic resistance (Taylor et al., 2002) and the stability of the resistance (Abu-El Samen et al., 2005). The use of various salts (Mills et al., 2005), foliar applications of phosphorous acid (Johnson et al., 2004) and the oomycete fungicides "zoxamide" and phosphite (Miller et al., 2006) have reduced symptoms of *P. erythroseptica* on tubers. Additional disease reduction technologies are still needed for organic markets and to deter the development of resistance to chemical fungicides. Tubers generally become infected in the field via stolons previously infected by germinating oospores (a thick-walled spore resulting from sexual recombination) but zoospores (motile, asexually produced spores) or encysted zoospores of the pathogen also can infect tuber eyes, lenticels and cracks and cuts that result from tuber harvesting operations--infection courts theoretically protectable using microbial antagonists. Therefore, 10 of our bacterial antagonists that reduce Fusarium dry rot, late blight, and/or sprouting in storage were assayed for efficacy against pink rot on tubers of cultivars Russet Burbank and Russet Norkotah (Schisler et al., 2009). Antagonist strains were grown in a semidefined liquid medium, diluted to ~3 x 108 cfu/ml, individually combined with zoospores of *P. erythroseptica,* and used to inoculate shallow puncture wounds on tubers. Data from full factorial experimental designs with 10 levels of antagonist, 2 levels of cultivar, and 2 levels of inoculum age after inducing zoospore liberation from sporangia indicated that all factors influenced the size of pink rot lesions that developed internally around wound sites *(P < 0.05).* In two different sets of experiments, *Enterobacter cloacae* strain S11:T:07 reduced lesion size more than the other antagonists (19% and 32% reduction versus the control) though

potato diseases and sprouting.

**6.3 Pink rot** 

As reviewed above, *Pseudomonas fluorescens* strains S11:P:12, P22:Y:05, and S22:T:04 and *Enterobacter cloacae* strain S11:T:07 have been documented as top strains to suppress four important storage potato maladies—dry rot, late blight, pink rot, and sprouting. These strains are known to differ from one another in their range of antibiotic production, substrate utilization, oxygen requirement, and growth temperature optima. They are also known to differ from one another in ability to inhibit sprouting or suppress disease on various potato cultivars and when incited by various pathogens. The variety of characteristics possessed by the individuals suggests that the successful strain mixtures are likely to be more resilient and more apt to provide individuals amenable to colonize potato wounds despite the variety of environments and pathogen strains encountered. Indeed, our previous experimental results have shown that certain strain pairs applied in combination allow greater dry rot suppression than do individual strains (Schisler et al., 1997). In subsequent laboratory and field trials, we observed that formulations containing multiple strains of our dry rot antagonists performed more consistently than individual strains did when subjected to 32 storage environments varying in potato cultivar, harvest year, potato washing procedure (microflora exposure), temperature, and storage time (Slininger et al., 2001). Successful biocontrol strain mixtures often contained both *Enterobacter cloacae* and *Pseudomonas fluorescens* strains. Several other research groups have reported that mixtures of strains can enhance and/or improve the consistency of biological control (among these, Pierson & Weller, 1994; Duffy & Weller, 1995; Duffy et al., 1996; Janisiewicz, 1996; Leeman et al., 1996; Guetsky et al., 2001; Krauss & Soberanis, 2001; Hwang & Benson, 2002; Schisler et al., 2005; Cruz et al., 2006). Thus, the formulation of strain mixtures has the potential to provide better, more consistent disease control than single strain formulations. Achieving consistent efficacy at each application represents a key advancement toward commercialization of any biocontrol product. However, despite the apparent advantages of applying strain mixes, the disadvantages for the manufacturer are capital costs, operation, maintenance, registration, and management of a different fermentation for each strain used in a mix. A potential way around this obstacle is to co-culture the strains together in one fermentor. To pursue the co-culture concept, we explored the level and consistency of pest control achievable on post-harvest potatoes with the four top multi-functional biological control agents *Pseudomonas fluorescens* strains S11:P:12, P22:Y:05, and S22:T:04 and *Enterobacter cloacae* S11:T:07 (Slininger et al., 2010b). The four bacteria were applied to potatoes in the following formats: a) as co-cultures of strains, i.e. multiple strains grown together in a single culture, b) as individual strains grown separately in pure cultures, and c) as blends of individual strains grown separately in pure cultures. Consistence of biocontrol efficacy and broad pest coverage, both major factors influencing the economics of a successful product, were addressed in this research. Treatments applied in both laboratory wounded potato bioassays and small pilot trials simulating commercial storage conditions were tested, as well as treatments challenged with dry rot, late blight, pink rot, and sprouting. Experiments were designed to analyze dry rot suppression versus all strain

Biological Control Agents for Suppression

enhanced efficacy, and enhanced consistence.

Wilbur, 1995; Slininger et al., 1997a, 1998).

**8. Production considerations** 

of Post-Harvest Diseases of Potatoes: Strategies on Discovery and Development 153

*Pseudomonas* sp. (Roberson & Firestone, 1992) and have been implicated as triggers to induced systemic resistance in host plants. It is believed that the EPS matrix slows the rate of water loss within the microenvironment, which enables the microbe additional time to make the necessary metabolic adjustments needed for survival. Relating to the current research, the co-culture of exopolysaccharide-producing *Paenibacillus* sp. with a *Pseudomonas* species has been reported to extend the shelf-life of the *Pseudomonas* for potential biopesticide or biofertilizer use (Kozyrovska et al., 2005). Thus, in addition to enhancing its biocontrol capacity, one of the benefits of our co-culture is that it includes an EPS-productive partner in *P. fluorescens* strain S11:P:12 that may protect bacteria against desiccation stress as they dry after application to tuber surfaces. The discovery of other mechanisms benefiting the function of the co-culture for consistent and efficacious biological control will likely be among the objectives of future research. Meanwhile, the advantages of co-culturing are compelling and spur on development efforts: economical production of multiple strains in one culture, broad disease spectrum, beneficial interactions of strains, desiccation sheltering,

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-

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 performance of the final population in suppressing dry rot disease.

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

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 *Pseudomonas* sp. (Roberson & Firestone, 1992) and have been implicated as triggers to induced systemic resistance in host plants. It is believed that the EPS matrix slows the rate of water loss within the microenvironment, which enables the microbe additional time to make the necessary metabolic adjustments needed for survival. Relating to the current research, the co-culture of exopolysaccharide-producing *Paenibacillus* sp. with a *Pseudomonas* species has been reported to extend the shelf-life of the *Pseudomonas* for potential biopesticide or biofertilizer use (Kozyrovska et al., 2005). Thus, in addition to enhancing its biocontrol capacity, one of the benefits of our co-culture is that it includes an EPS-productive partner in *P. fluorescens* strain S11:P:12 that may protect bacteria against desiccation stress as they dry after application to tuber surfaces. The discovery of other mechanisms benefiting the function of the co-culture for consistent and efficacious biological control will likely be among the objectives of future research. Meanwhile, the advantages of co-culturing are compelling and spur on development efforts: economical production of multiple strains in one culture, broad disease spectrum, beneficial interactions of strains, desiccation sheltering, enhanced efficacy, and enhanced consistence.
