**5. Bioautography as a screen for the presence of antibiotic production**

A variety of potential mechanisms have generally been proposed to be involved in the biological control of plant diseases, including antibiosis, induced disease resistance, competition, parasitism, and predation. Works by Fravel (1988), Huang (1991), Loper & Buyer (1991), Schisler (1997), and Wilson et al. (1994) are useful starting points for information on mechanisms of biological control and microbial interactions potentially of relevance to dry rot disease development. Antibiosis, induced disease resistance, and competition are all possible mechanisms of control for any of our most effective strains of bacteria. However, with regard to mode of action, our studies have focused on the influence of microbial metabolites on *G. pulicaris.* In petri plate assays against the dry rot pathogen, *G. pulicaris,* our 18 bacterial cultures showed varying degrees of inhibition of fungal growth. When extracts from liquid cultures were tested by thin-layer chromatography-bioautography (TLC-BA), a useful technique which correlates antimicrobial activity with the presence of antibiotics (Lazarovits et al., 1982; Homma & Suzui, 1989), all of the cultures tested were shown to produce at least one compound which inhibited the growth of *G. pulicaris* (Burkhead et al., 1995).

Antifungal metabolites from *Enterobacter cloacae* strain S11:T:07 NRRL B-21050, which was highly ranked by the 2DLCF procedure (Table 1, Figure 2), have been isolated from Sabouraud maltose broth culture and identified as phenylacetic acid (PAA), indoleacetic acid (IAA), tyrosol (TSL), and tyrosol acetate, which are recognized to be derived from aromatic amino acids (Burkhead et al., 1998; Slininger et al., 2004). Consequently in later experiments when these compounds were assayed in cultures of strain S11:T:07 (B-21050) grown in three different growth media, it was not a surprise to learn that relative composition of the antifungal compounds produced varied as the culture nutrition, especially amino acid composition, was varied. Antifungal and sprout regulatory bioactivities of these compounds (alone and in combination) were further investigated using our wounded potato assay of dry rot suppressiveness and a cored potato eye assay of sprout inhibition. Assay results showed the antifungal activity of IAA, PAA, and TSL to suppress dry rot infection of wounded potatoes and indicated optimal efficacy when all three metabolites were applied in combination. Furthermore, dosages of IAA resulting in disease suppression, also resulted in sprout inhibition. These results suggest the potential for designing culture production and formulation conditions to achieve a dual purpose biological control agent able to suppress both dry rot and sprouting (Slininger et al., 2004).

### **6. Expanding the available market with broad spectrum biological control**

The observation of many antifungal compounds per each dry rot suppressive isolate and the finding of diverse functional activities ranging from antifungal antibiotics to plant regulatory hormones of isolated antifungal compounds suggested the fruitfulness of exploring the spectrum of use as a means of improving the market draw for our biological control agents.

#### **6.1 Sprout inhibition**

148 Fungicides for Plant and Animal Diseases

multiple cultivars could be selected using the dimensionless relative performance index concept. The ability of biocontrol agents to solve multiple pest control problems is another potential screening dimension. For example, our dry rot antagonistic bacteria have also been shown to be able to suppress late blight (Slininger et al., 2007), pink rot (Schisler et al. 2009), and sprouting of stored potatoes (Slininger et al., 2000, 2003). The ability to expand the market to multiple pest control applications is expected to enhance commercial development potential of a given biocontrol product and is a recurring theme influencing the progression of our research as will be discussed at various points later in this account.

**5. Bioautography as a screen for the presence of antibiotic production** 

compound which inhibited the growth of *G. pulicaris* (Burkhead et al., 1995).

A variety of potential mechanisms have generally been proposed to be involved in the biological control of plant diseases, including antibiosis, induced disease resistance, competition, parasitism, and predation. Works by Fravel (1988), Huang (1991), Loper & Buyer (1991), Schisler (1997), and Wilson et al. (1994) are useful starting points for information on mechanisms of biological control and microbial interactions potentially of relevance to dry rot disease development. Antibiosis, induced disease resistance, and competition are all possible mechanisms of control for any of our most effective strains of bacteria. However, with regard to mode of action, our studies have focused on the influence of microbial metabolites on *G. pulicaris.* In petri plate assays against the dry rot pathogen, *G. pulicaris,* our 18 bacterial cultures showed varying degrees of inhibition of fungal growth. When extracts from liquid cultures were tested by thin-layer chromatography-bioautography (TLC-BA), a useful technique which correlates antimicrobial activity with the presence of antibiotics (Lazarovits et al., 1982; Homma & Suzui, 1989), all of the cultures tested were shown to produce at least one

Antifungal metabolites from *Enterobacter cloacae* strain S11:T:07 NRRL B-21050, which was highly ranked by the 2DLCF procedure (Table 1, Figure 2), have been isolated from Sabouraud maltose broth culture and identified as phenylacetic acid (PAA), indoleacetic acid (IAA), tyrosol (TSL), and tyrosol acetate, which are recognized to be derived from aromatic amino acids (Burkhead et al., 1998; Slininger et al., 2004). Consequently in later experiments when these compounds were assayed in cultures of strain S11:T:07 (B-21050) grown in three different growth media, it was not a surprise to learn that relative composition of the antifungal compounds produced varied as the culture nutrition, especially amino acid composition, was varied. Antifungal and sprout regulatory bioactivities of these compounds (alone and in combination) were further investigated using our wounded potato assay of dry rot suppressiveness and a cored potato eye assay of sprout inhibition. Assay results showed the antifungal activity of IAA, PAA, and TSL to suppress dry rot infection of wounded potatoes and indicated optimal efficacy when all three metabolites were applied in combination. Furthermore, dosages of IAA resulting in disease suppression, also resulted in sprout inhibition. These results suggest the potential for designing culture production and formulation conditions to achieve a dual purpose biological control agent able to suppress both dry rot and sprouting (Slininger et al., 2004).

**6. Expanding the available market with broad spectrum biological control** 

The observation of many antifungal compounds per each dry rot suppressive isolate and the finding of diverse functional activities ranging from antifungal antibiotics to plant Current practices for reducing sprouting in storage could also benefit from microbial alternatives. Because of processing demands, over 54% of the annual potato harvest must be stored at 7º to 13ºC, a temperature range above that needed for ideal sprout control (ASAE, 1990). Chemical sprout inhibitors are applied to over 50% of the potato harvest to extend storage time. The potato industry has become very dependent on CIPC (1-methylethyl-3 chlorophenylcarbamate) as the most efficient sprout inhibitor with fewest detrimental sideeffects on process potato quality (Lewis et al., 1996). However, recently, the tolerance for residues of CIPC has been reduced to 30 mg/kg (EPA 738-R-96-023, 1996) because of CIPC's persistence in the environment and potato tissue and concerns about its toxicity (Mondy et al., 1992). In the U.S.A., CIPC is the only synthetic chemical registered for post-harvest sprout control of stored potatoes, and it is the most widely used sprout inhibitor worldwide. Due to environmental and health safety concerns, the use of CIPC has become more restricted--opening a potential market for alternative sprout control methods. Consequently, six of our bacteria strains, exhibiting superior dry rot suppressiveness in previous research, were grown in two different liquid culture media and sprayed on Russet Burbank potatoes to assay sprout suppresiveness (Slininger et al., 2000, 2003). In growth chamber and pilot experiments repeated at two storage sites in two successive years, all six isolates demonstrated significant sprout control capabilities when applied after growth on at least one of the culture media supplied. Of the six strains tested, *Pseudomonas fluorescens* S11:P:12 (NRRL B-21133) and two strains of *Enterobacter* sp., S11:T:07 (NRRL B-21050) and S11:P:08 (NRRL B-21132), exhibited highest relative performance levels with sprout control being statistically similar to that of 16.6 ppm CIPC thermal fog after 4-5 months storage.

#### **6.2 Late blight**

Several of our top six dry rot suppressive strains have now also been found to significantly reduce late blight infection of stored potatoes (Slininger et al., 2007). Consistent with our observations of indoleacetic acid (IAA) as a major antifungal product produced by one of our dry rot suppressive strains (Slininger et al., 2004), Martiniez Noel et al. (2001) also previously showed that IAA attenuates disease severity in potato-*Phytopthora infestans* interactions and inhibits pathogen growth *in vitro*. *Phytopthora infestans*, the causative agent of the potato late blight disease, infects tubers through eyes or wounds, primarily via zoospores washed into soil from sporangia on infected leaves. Harvested tubers can become infected during washing (Fairclough et al., 1997) and during storage and handling (Lambert et al., 1998). *Phytopthora infestans* is considered to be the most significant pathogen of the crop worldwide (Fry et al., 2001) and historically was the cause of the Great Potato Famine of the late 1840's. The introduction of US-8 genotypes of *P. infestans* has coincided with an increase in severity of potato late blight in North America. As alternatives to chemical fungicides, our 18 bacterial strains patented as biological control agents of both sprouting and Fusarium dry rot were cultivated in 3 liquid media and screened in wounded potato bioassays for their ability to suppress late blight incited by *P. infestans* (US-8, mating type A2) (Slininger et al., 2007). Washed or unwashed stationary-phase bacteria were mixed with

Biological Control Agents for Suppression

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

performance.

of Post-Harvest Diseases of Potatoes: Strategies on Discovery and Development 151

*Pseudomonas fluorescens* S11:P:14, *Pseudomonas* sp. S22:T:04, and *Enterobacter* sp S11:P:08 also significantly reduced disease. Lesion size was greater on Russet Norkotah than Russet Burbank tubers (42.3 and 26.5 mm, respectively), but cultivar did not influence antagonist

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

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 potato diseases and sprouting.

#### **6.3 Pink rot**

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 *Pseudomonas fluorescens* S11:P:14, *Pseudomonas* sp. S22:T:04, and *Enterobacter* sp S11:P:08 also significantly reduced disease. Lesion size was greater on Russet Norkotah than Russet Burbank tubers (42.3 and 26.5 mm, respectively), but cultivar did not influence antagonist performance.
