**Biological Control Agents for Suppression of Post-Harvest Diseases of Potatoes: Strategies on Discovery and Development**

Patricia J. Slininger and David A. Schisler *National Center for Agricultural Utilization Research, United States Department of Agriculture, Agricultural Research Service, Peoria, IL United States of America* 

#### **1. Introduction**

As used in plant pathology, the term "biological control" or its short form "biocontrol" commonly refers to the decrease in the inoculum or the disease-producing activity of a pathogen accomplished through one or more organisms, including the host plant but excluding man (Baker, 1987). Biological control of plant pathogens naturally occurs at some level in all agricultural ecosystems, sometimes to a degree where symptoms of disease are noticeably reduced. Thousands of potential microbial biocontrol agents have been isolated from agricultural fields and crops during research over the last 80 years, yet only a few are in commercial use. Recently, public health and safety concerns about the environmental impact of chemical pesticides have led to consideration of biological control as a natural approach to maintaining crop health. Despite environmental incentives and strong research efforts, commercialization of biocontrol agents has been slow to evolve. The momentum of the chemical industry is difficult to shift, and fermentation processes tend to be more expensive to operate than synthetic chemical processes. Yet there is a demand for biological control products, especially in the organic and agricultural niche markets, where there is no efficient chemical competitor. Indeed, the tide has been turning and a recent story in Chemical and Engineering News (Reisch, 2011) has indicated that during the last decade, the growth in sales of biological pest control agents has significantly outpaced that of chemicals. However, given this market demand, the fundamental methods of economical large-scale production and application of biological control agents are still lacking and need to be developed. Many aspects of biocontrol agent production and development represent untrodden territory in the progression of industrial fermentation technology beyond its well-established food and pharmaceuticals niche. Distinguishing them from traditional fermentation products, biocontrol agents must not only be produced in high yield but must also meet the following quality criteria: high (near 100%) retention of cell viability with maintenance of crop compatibility and consistent bioefficacy during several months of storage.

Biological Control Agents for Suppression

true for many post-harvest pathosystems.

of Post-Harvest Diseases of Potatoes: Strategies on Discovery and Development 143

introduced antagonists, availability of few or no control options, and causative of significant economic loss to agriculture. Our experience on discovery and development of biological control agents first began with the need to find an alternative to thiabendazole (TBZ) for the biological control of Fusarium dry rot, an important post-harvest disease of potatoes. Dry rot is caused primarily by *Gibberella pulicaris* (Fr.:Fr.) Sacc. (anamorph: *Fusarium sambucinum* Fuckel) (Boyd, 1972). The fungus is a serious pathogen in potato tuber storages and can produce trichothecene toxins (Desjardins & Plattner, 1989) implicated in mycotoxicosis of humans and animals. Yield losses attributed to dry rot in storage range from 6 to 25% with up to 60% of tubers affected in some cases (Secor & Salas, 2001). Measures for controlling this disease in storage are limited. Resistance to TBZ, the only chemical registered for postharvest use on tubers for human consumption, is now widespread among strains of *G. pulicaris* (Desjardins et al., 1993; Hanson et al., 1996; Kawchuk et al., 1994; Secor et al., 1994). High levels of resistance to Fusarium dry rot in potato cultivars and breeders' selections are not apparent (Pawlak et al., 1987) and all commonly grown potato cultivars are susceptible (Reiners & Petzoldt, 2004). Therefore, the potential for damage is high enough to justify the economic risk of developing a biological control agent for prevention of dry rot disease losses. A major weakness of the etiology of this pathogen is that it requires a wound in order to infect, and tubers are able to heal wounds in less than 2 weeks in storage. Additionally, the pathogen operates in an environment that is favorable to introduced antagonists in that tuber storage temperatures are uniform and relative humidities are high (>90%), a feature

**4. Discovery of biocontrol agents amenable to commercial production** 

useful agents based on the challenges of manufacturing and delivery.

**4.1 Rapid isolation from large populations via enrichment techniques** 

Main objectives driving the development of our techniques to discover beneficial biological control agents for dry rot suppression involved two phases: (1) rapid screening of large numbers of microbes using enrichment techniques to concentrate desirable populations and a crop-relevant bioassay to identify useful biological control agents; (2) rating potentially

Ideally biological control agent isolation should begin in areas where biological control is naturally occurring in the field, as opposed to areas where it is not. Evaluating a maximal number of putative biocontrol agents increases the chance of discovering an effective strain. Isolating prospective biocontrol agents from appropriate tissues and under appropriate environmental conditions helps to insure that the microbial antagonists isolated will be well adapted to survival and activity on the specific tissues requiring protection. Application of these concepts resulted in our rapid isolation of 18 putative biological control agents for suppression of Fusarium dry rot. The steps of our method are illustrated in Figure 1 (Schisler & Slininger, 1994). Specifically, gamma irradiation-sterilized field soil samples were first enriched with potato tuber periderm, inoculated with a small amount of field soil obtained from potato fields with low dry rot disease incidence, and incubated for 1 week at 15°C. The microorganisms most adept at rapid growth on the nutrients found in potato periderm and at wound sites would make up the majority of microbes in each recolonized soil sample. Next, conidia of *G. pulicaris* were added to the microbially recolonized soils, and 2 days later, aqueous soil pastes of each soil were applied to wounded potato tubers to initiate a realistic disease bioassay. After incubation 4 weeks at 15°C, tubers were scored for

This article will focus on the control of post-harvest fungal pathogens, which present unique opportunities but also challenges. Though accurately determining the extent of losses is difficult and few reports are available, it has been estimated that post-harvest decay accounts for an approximate 25% loss of fresh commodities (USDA, 1965). Biological control using microbial antagonists can be an appropriate tool for managing post-harvest disease problems, especially in crops which are stored under controlled temperatures and high relative humidities. Such controlled storage environments represent a luxury not found when attempting to introduce microbial biological control agents into the comparatively harsh, variable environments found at the infection courts of fungal pathogens of field-grown plants. In recent years, a considerable research attention has focused on biologically controlling rots of fruits post harvest (Janisiewicz, 1988, 1991, 2002). In this chapter, research examples will be reviewed to illustrate the challenges and strategies of developing processes to manufacture and deliver biological agents for post-harvest potato disease control. Concepts to be covered will include the following: market opportunities, choosing pathosystems for biological control, enrichment techniques to enhance new strain discovery, strategies for ranking strains for commercial suitability, mode of action, production considerations, market-broadening functionality, co-cultivation of strains as the next generation biocontrol product, highthroughput screen concept for optimizing biocontrol agent performance from production to delivery, remaining knowledge gaps, and future investigations.
