Chapter 10 Quo Vadis of Biological Control of Postharvest Diseases

Chapter 10 Quo Vadis of Biological Control of Postharvest Diseases Wojciech J. Janisiewicz Abstract Research on Biological Control of Postharvest Diseases (BCPD) has been conducted for over two decades and successes, present and future direction are being discussed. The BCPB has been accepted by the fruit and vegetable industry as the stand alone or in combination with other commercial treatments, depending on fruit and vegetable. BioSave has been on the market since 1996, and its use is expanding to control more postharvest diseases of fruits and vegetables. World-wide efforts in developing BCPD resulted in the registration of more products recently. The number of scientific publications is also increasing steadily. As postharvest biocontrol products are coming to the market, their anticipated limitations are become apparent, and much of the current research is focused on addressing these limitations. Combining antagonists with various substances Generally Regarded as Safe (GRASS), such as sodium bicarbonate, calcium chloride, diluted ethanol, or with physical treatments such as heat, or UV irradiation are typical examples of approaches being used. A mixture of two compatible biocontrol agents often showed an additive or synergistic effect in controlling fruit decays. Biocontrol agents developed for the control of fruit decays have also been shown to inhibit growth of foodborne human pathogens. This aspect gains in importance as new outbreaks are reported with increasing frequency, and fresh cut fruit and vegetables are particularly vulnerable to colonization by foodborne pathogens. Biocontrol agents can also control decays originating from wounds made during mechanical harvesting of fruits. Currently, available biocontrol products were developed for the control of decays originating from the infection of fruit wounds, but the next greatest challenge for BCPD research is the development of the next generation of biocontrol products that control latent infections. Many important diseases of temperate, subtropical and tropical fruit, including those caused by Monillinia spp. and Colletotrichum spp.., originate from these infections in the orchard and cause decay on fruits in storage. This research requires broadening the pool of microorganisms W.J. Janisiewicz Agricultural Research Service, U.S. Department of Agriculture, Appalachian Fruit Research Station, 2217, Wiltshire Road, Kearneysville, WV, 25430, USA e-mail: wojciech.janisiewicz@ars.usda.gov D. Prusky and M.L. Gullino (eds.), Postharvest Pathology, Plant Pathology in the 21st Century, Vol. 2, DOI 10.1007/978-1-4020-8930-5_10, Springer Science + Business Media B.V. 2010 137

138 W.J. Janisiewicz screened for biological control activity to include, in addition to those occurring naturally on fruit, microorganisms from different plants and plant parts, as well as microorganisms from different habitats. Some programs are already focusing in this direction, and there is great hope and optimism that at the next ISPP Congress in 2013 in Beijing, we will have reports on the significant progress in this area. Keywords Biocontrol, Biocontrol products, Emerging biocontrol, Postharvest decay 10.1 Historical Perspective This is the fourth consecutive evening session on Biological Control of Postharvest Diseases (BCPD) at the ICPP and it is axiomatic that, after more than two decades of research on BCPD, we should reflect on the direction of the current research and what we can expect in the foreseeable future. A SCOPUS survey of the last 10 years of research on BCPD indicates an increasing number of peer-review publications from 19 in 1998 to 54 in 2008 (a projected number based on 38 publications by the end of August, 2008). Most of the research has been conducted in Europe (mainly Belgium, Italy and Spain, and Sweden), United States, Israel, South Africa, and more recently in China. Many smaller programs developing in other countries also contribute significantly to progress in this field, and further indicate the growing interest in BCPD. The expansion of this research began with the report by Pusey and Wilson (1984) on the successful control of brown rot of peach and other stone fruits caused by Monilinia fructicola after harvest using soil isolated Bacillus subtilis (strain B3). Postharvest application attempts were made after the field application of this bacterium to peach trees from bloom to harvest failed to control this disease. The authors concluded that controlled environmental conditions in the postharvest system provide a more stable environment for the antagonist resulting in a high level of disease control (Wilson and Pusey 1985). Results from pilot tests on the control of brown rot of peach, conducted in commercial packinghouses, were encouraging, but this antagonist was never commercialized (Pusey et al. 1988). The discovery of bacterial and yeast antagonists effective against various postharvest diseases of pome fruits among resident microflora of apple and pear provided a new source of antagonists. It also made us realize that potential antagonists on the surfaces of these fruits have long been consumed by humans without any apparent adverse effects (Janisiewicz 1987). The next milestone was the registration by the United States Environmental Protection agency (EPA) and commercialization of the first two biocontrol agents: a yeast Candida oleophila, used in Aspire (Droby et al. 1998), and a saprophytic strain of Pseudomonas syringae, used in BioSave (Janisiewicz and Jeffers 1997) in 1995. In this biotechnology era, each year seems to be a millennium, and the success of new commercial biocontrol products is often measured by their life span on the market. Three years of commercial use seems to be the breaking point for many biocontrol products. In

10 Quo Vadis of Biological Control of Postharvest Diseases 139 this context, the last milestone was achieved in 2006, the 10-year anniversary of the large scale commercial use of BioSave. There have been many important advances in BCPD during the past decade, especially in the area of mechanisms of biocontrol, manipulation of antagonist physiology for the benefit of biocontrol, development of formulations, and integration with other control methods. Only time will indicate the full impact of these advances on the increased use of BCPB. 10.2 Biocontrol Products Aspire and BioSave lead the way in commercial application of biocontrol agents to fruit. Other products such as YieldPlus based on Cryptococcus albidus, Avogreen based on Bacillus subtilis, and Shemer based on Metschnikowia fructicola are also on the market in various countries. Aspire has been registered in the United States for postharvest application to citrus and pome fruits. This product was taken off the market 3 years after its large scale commercial introduction. BioSave (two strains of P. syringae) was originally registered for postharvest application to pome and citrus fruits, and this was later extended to cherries, potatoes, and more recently to sweet potatoes. YieldPlus was developed in South Africa for postharvest application to pome fruits but the success of this product is largely unknown and there is no published literature or information available to determine extent of its use. Avogreen has been used for control of postharvest disease of avocado. Its use has been limited (L. Korsten, personal communication), possibly due to inconsistent results. More recently, Shemer was registered in Israel for both pre- and postharvest application on various fruits and vegetables including apricot, citrus, grapes, peach, pepper, strawberry and sweet potato. There are three more products coming to the market: Candifruit based on Candida sake, developed in Spain; Boni-Protect, based on Aureobasidium pullulans, developed in Germany and NEXY, based on Candida oleophila, developed in Belgium. All of these products have been registered for control of postharvest diseases of pome fruits. These new products are further testimony that increasing interest in BCPD is not just a matter of scientific curiosity but have resulted in diligent efforts to implement this approach. Gradual removal of the major regulatory barriers to registration of antagonists for BCPD in different countries is also very encouraging. 10.3 Postharvest System BCPD now encompasses the use of antagonists to control postharvest diseases of fruits, vegetables and grains. Postharvest biocontrol of flower diseases is another area awaiting exploration. Early attempts to use a metabolite (pyrrolnitrin) of a bacterial antagonist to control cut rose flower infections were very encouraging (Hammer et al. 1993) but little else has been done in this field. Biocontrol of grain

140 W.J. Janisiewicz spoilage during storage in silos has made significant advances and appears to be on the way to commercialization (Peterson and Schnurer 1995; Peterson et al. 1999; Druvefors 2004). Examples from commercially used biocontrol products indicate that biocontrol agents developed for BCPD on one commodity could also be effective against the same or different pathogens on other commodities. The activity of biocontrol agents is not as universal as fungicides but is less specialized than originally anticipated. Broadening the application of biocontrol agents is a good strategy for commercial success. It not only makes the product more profitable and allows for a quicker return on the investment made in the commercialization of the product, but it also allows a buffer in the fluctuation in the market due to registration of new fungicides or other alternative products. As postharvest biocontrol products are coming to the market, their anticipated limitations are elucidated and much of the current research is focused on addressing these limitations. The most commonly used approach is combining antagonists with various substances Generally Regarded as Safe (GRASS), sodium bicarbonate (SBC), calcium chloride, or ethanol (Bertolini 2008; Karabulut et al. 2003). These combinations both reduced the fluctuation and increased the level of decay control. Combining antagonists with other alternatives to fungicide treatments also showed good results, but its implementation often requires modifying currently used postharvest practices or adds substantially to the cost of the treatment. For example, a heat treatment of apples with hot air at 38 C for 4 days or oranges at 30 C for 1 day after harvest in combination with antagonists gave superior control (including eradicative activity) of blue mold and gray mold, respectively, compared to the individual treatments (Huang et al. 1995; Leverentz et al. 2000, Leverentz et al. 2003c). However, this approach will require adding heating equipment and changing the temperature in of storage rooms. Some packinghouses use high temperatures to sanitize empty bins and storage rooms, but even this practice is still very limited. It is also well established that the proper selection of the combination of two antagonists can provide superior decay control to either antagonists applied individually (Calvo et al. 2003; Janisiewicz and Bors 1995). Although using this approach is very attractive, it doubles the cost of registration because each antagonist must be registered separately. This problem could be eliminated if biocontrol products currently on the market could be combined resulting in additive or synergistic effects. There is increasing consensus that, in the future, non-fungicidal control will rely on the combination of various treatments with biocontrol. This combination of treatments is similarly to the hurdle concept developed by Leistner in 1978 for the reduction of food contamination with foodborne pathogens, where each additional treatment further reduces the possibility of food contamination (Leistner 1978, 2000). 10.4 Challenges of Latent Infection All biocontrol agents currently registered for postharvest application control fruit decays originating from wound infections made during or after harvest (see review by Janisiewicz and Korsten 2002). Although for some fruits, such as pome or citrus

10 Quo Vadis of Biological Control of Postharvest Diseases 141 (depending on the region) fruits, wounds are the main court of entry for postharvest decay causing fungi, many postharvest decays of stone fruits and subtropical fruits develop in storage from latent infections occurring in the orchard. These infections are difficult to control because the intimate relationship of the pathogen with the host has been already established, and melanized appressoria often formed by these fungi on fruit surface are very resistant to environmental factors and penetration by fungicides. Control of latent infection with microbial antagonists is the next big challenge to BCPD. Earlier attempts to reduce decay originating from latent infections on mango fruit indicate that microbial antagonists could be effective against this type of infection (Koomen and Jeffries 1993), however this work did not continue beyond initial reports, suggesting difficulties in making further progress. New screening and fruit testing approaches must be developed to address latent infection. One of the approaches used in our laboratory employs in vitro screening for biocontrol activity against M. fructicola and Colletotrichum accutatum on crystalline cellulose membranes impregnated with waxes isolated from apple, plum or nectarine fruit. Both fungi produce melanized appressoria on these membranes when inoculated as an aqueous suspension of the conidia, and can infect fruit from these appressoria if the membrane is placed on the fruit. Bacteria and yeast can be applied to the membrane surface with the appressoria, and those showing growth around appressoria and/or reducing fruit infection from these membranes are selected for further testing directly on fruit. Our current challenge is to make this procedure more quantitative. Unlike with wound infecting fungi, where screening for antagonists was independent of the mechanism of biocontrol, screening against latent infections could benefit from focusing on organisms exhibiting mechanisms of biocontrol which could most likely succeed against the melanized structures of the pathogen. The primary candidates are microorganisms producing volatile substances and enzymes capable of degrading the melanized structure of the fungi. Predacious yeasts may also be useful. Antifungal volatiles produced by the antagonist Muscodor albus, discovered as an endophyte of Cinnamomun zeylanicium in Honduras, have been shown recently to control major postharvest pathogens on a variety of fruits (Strobel 2006; Strobel et al. 2001; Mercier and Jiménez 2004; Mercier and Smilanick 2005). Focusing the search on this mechanism could greatly enhance opportunities of finding antagonists with volatiles capable of penetrating melanized structures of the fungi. This example also indicates that microflora of exotic plants, especially endophytes, is an untappped resource and should be explored for their biocontrol potential. With the exception of grapes, pome, citrus and several other fruits, information about resident microflora of fruit and fruit tree plants is very limited and their potential for BCPD is largely unexplored. 10.5 Biological Control Mechanism Studies of the ecology and physiology of antagonists were the main driving forces during initial development of BCPD that lead to commercial products, but research on the mechanisms of biological control (MBC), that have been lagging behind, has

142 W.J. Janisiewicz made significant progress especially during the past decade. This subject is covered in Chapter 12 by R. Castoria and Sandra A.I. Wright. Here, I would like to point out a few factors that have contributed to this progress. More focus and resources have been put into studying mechanisms of BCPD after the first commercial products began to be marketed on a large scale in late 1990s. The limitations to BCPD have become a reality in commercial settings and the knowledge of the MBC has been look upon as one of the ways that may address some of these limitations. Advances in molecular biology have allowed us to address, in a more direct manner, questions about the role of some putative MBC e.g. lytic enzymes or reactive oxygen species (ROS) (Castoria et al. 2005; Chan et al. 2007; Friel et al. 2007; Macarisin et al. 2007; Massart and Jijakli 2006; Xu and Tian 2008). In most cases more than one mechanism of biocontrol has been implicated and the significance of different mechanisms of biocontrol still needs to be established. Development of a bacterial and yeast model systems, where genes for different MBC traits can be expressed, and where the transformants can be tested on fruit for biocontrol potential, could be very helpful in many cases. A good examples of what could be accomplished in the fruit system is work with, Saccharomyces cerevisiae and Picha pastoris, non-antagonistic yeasts which were transformed with genes coding for antifungal peptides cercopin (defensin from insects) and Psd1 (defensin from pea seeds), respectively. These transformants greatly reduced postharvest decays on tomato and pome fruits (Jones and Prusky 2002; Janisiewicz et al. 2008). There are genes currently available that could be tested in such a system, e.g. genes coding for antifungal pyrolnitrin, which were isolated from Pseudomonas fluorescent, and also produced by Pseudomonas cepacia (now Burkholderia cepacia), an excellent biocontrol agent against various postharvest decays on pome, stone and citrus fruits (Janisiewicz and Roitman 1988; Hammer et al. 1997). Other good candidates are genes responsible for the production of various lytic enzymes, ROS or siderophores. Eventually, this approach may lead to improvement of existing antagonists or the development of new ones (as with cercopin and Psd1 defensins), however for this to have a practical outcome, the hurdles of safety and public acceptance will have to be overcome (Janisiewicz 1998). The discovery of a very effective antagonist producing antifungal volatiles indicates that a single MBC can be sufficient to provide adequate control of a fruit decay. This also justifies screening for a single MBC, if an effective mechanism can be identified, and makes in vitro screening more meaningful and effective because it allows the screening of vast numbers of organisms in a short period of time before resorting to more expensive and time consuming tests on fruit. 10.6 Emerging New Areas in Postharvest Biocontrol During the past decade, more emphasis has been put on the safety of fruits and vegetables as an increasing number of outbreaks with foodborne human diseases are reported each year following consumption of various fruit and vegetables. The situation is worsened by growing consumption of fresh-cut fruits and vegetables,

10 Quo Vadis of Biological Control of Postharvest Diseases 143 which provide a conducive environment for the growth of various bacterial human pathogens. This also creates an opportunity for the use of microbial antagonists to combat these foodborne pathogens. For example, Pseudomonas syringae used in Bio-Save can prevent the growth of E. coli in apple wounds (Janisiewicz et al. 1999), and several other biocontrol agents, e.g. Gluconobacter asaii, Discofaerina fagi, or Metschnikowia pulcherrima, developed for control of postharvest decays, not only prevented growth but even reduced populations of the foodborne pathogens Listeria monocytogenes and Salmonella enterica sv. Poona on fresh cut apples (Leverentz et al. 2006). The concept and the potential for using various microorganisms against human foodborne pathogens on minimally processed fruits and vegetables has been discussed (Leverentz et al. 2003b). Lactic acid bacteria (LAB) such as Lactobacillus plantarum, Lactococcus lactis, Leuconostoc sp., or Weissella sp., occur frequently on fruits and vegetables and many strains of these LAB prevented growth or even reduced populations of the major foodborne pathogens on apples and lettuce (Trias et al. 2008). Lytic bacteriophages can be very effective in reducing populations of foodborne pathogens (Leverentz et al. 2001). They can be combined with other bacterial or yeast antagonist or the bacteriocin, niacin, to achieve the targeted level of a 6 log unit reduction in populations of the foodborne pathogens (Leverentz et al. 2003a, Fig. 10.1). In 2005, the United States Environmental Protection Agency approved, for the first time, the use of bacteriophages as a food additive to reduce contamination of foods with foodborne pathogens. It is not incidental that the control of foodborne pathogens has recently been the topic of various sessions, including the Plenary Session, at the APS Annual Meetings, as the growth of these pathogens can be intertwined with the growth of plant pathogens. Recent studies indicate that a diseased tissue can be more prone to colonization by foodborne human pathogens than the healthy tissue. For example, apple tissue infected with Glomerella cingulata, causing bitter rot, becomes less acidic allowing colonization by L. monocytogenes (Conway et al. 2000). Labor shortages, especially in the United States and other developed countries, have increased the demand for mechanical harvesting of fruits and vegetables. Significant progress has been made during the past two decades and some fruits are currently harvested mechanically, not only for processing, but also for the fresh market. Despite these major advances, fruit wounding is still a problem and is the major limitation for developing this harvesting technology. Since the majority of postharvest pathogens of temperate fruit crops infect fruits through wounds which results in decay in storage, this presents an ideal opportunity for the use of biological control. Considering that the most successful BCPD to date is against wound invading pathogens, this area is worth of exploring (Janisiewicz and Korsten 2002). The first attempt to biologically control decay originating from wounds made during mechanical harvesting suggests that this approach is feasible (Janisiewicz and Peterson 2004). Stem loss (stempulls) on mechanically harvested apples range from 20 57%, depending on cultivar. If a portion of apple skin is removed together with the stem, flesh tissue is exposed, creating a potential entry site for the pathogen. The P. syringae (ESC-11) strain that is used in BioSave reduced blue mold decay on mechanically harvested Empire apples with stempulls from 41% to 3.3% and this antagonist completely eliminated decay on other, less susceptible, cultivars

144 W.J. Janisiewicz Lm Lm+Phage Lm+G.asaii Lm+Phage+G.asaii logcfu/plug 8 7 6 5 4 3 2 1 0 2 5 7 Storage Time (d) Treatment 0 2 5 7 L LP LG LPG 2.64a 1 z 2 2.74az 2.49ay 2.79ax 4.45ay 3.01bz 4.01ax 3.07bx 7.06ax 5.54y 3.95cx 2.50dxy 7.474ax 6.38bx 3.65cx 1.61dy 1 Treatment means within Da y with different a, b, c, d letters are different at the 0.05 significance level. 2 Da y means within Treatment with different x, y, z letters are different at the 0.05 significance level. Fig. 10.1 Recovery of Listeria monocytogens from honeydew wedges treated with the pathogen (10 4 CFU/mL) alone (L) or in combination with phage (LP), Gluconobacter asaii (LG). or a combination of the two (LPG) and stored at 10 C over 7 days (Hong Y, Leverentz B, Coway WS, Janisiewicz WJ, Abadias M, Camp MJ, unpublished) (Table. 10.1). A similar situation may occur with harvested sweet cherries where the first mechanical prototypes are already being used commercially and all of the fruit is harvested without stems (Peterson and Wolford 2001). Commercially mechanically harvested blueberry may also provide an opportunity for biocontrol, and the development of blackberry mechanical harvesters has made significant progress recently (Peterson et al. 1997; Peterson and Takeda 2003; Takeda et al. 2008). 10.7 Conclusions We are entering into a new era in BCPD with expanding scientific interest and new products coming to the market. Commercial use of the pioneering products indicates that the fruit and vegetable industry will accept biological control, as long as

10 Quo Vadis of Biological Control of Postharvest Diseases 145 Table 10.1 Development of blue mold in the stem cavity area of mechanically harvested apples with and without stems (stempulls) after inoculation with 25 µl of Penicillium expansum suspension (5 10 5 conidia/ml) alone or in combination with the antagonist Pseudomonas syringae (ESC- 11). Fruit were stored at 1 C for 2 months (Janisiewicz WJ, Peterson DL, unpublished) it provides adequate control. The persistent efforts in many programs focusing on development of new biocontrol agents and/or studying mechanisms of biocontrol are beginning to pay off. Regulatory agencies in an increasing number of countries are accepting biocontrol products as safe alternative to fungicide treatments. Currently, most of the biocontrol products are registered in individual countries, but expanding registration to other countries would facilitate greater use of the products and possibly promote the use of mixtures of some products if more than one product is registered in a country, as combinations of compatible antagonists often provided improved decay control compared to individual antagonists used alone. Examples presented earlier indicates that greater use of BCPD can be achieved by expanding its use to new commodities, new applications, and by integrating biocontrol with other treatments. Current successes with biocontrol of decays originating from fruit wounds gives us optimism that the next great challenge, the control of latent infections, also can be achieved. This, most likely, will require broadening the pool of microorganisms screened for biocontrol activity to include epiphytes and endophytes from different plants and plant parts, as well as microorganisms from different habitats. With research in this direction already under way, there is great hope and optimism that at the next ISPP Congress in 2013 in Beijing we will have reports that the next generation of biocontrol agents are capable of controlling latent infections of fruit.

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148 W.J. Janisiewicz Pusey PL, Hotchkiss MW, Dulmage HT, Baumgardner RA, Zher EI, Reilly CC, Wilson CL (1988) Pilot test for commercial production and application of Bacillus subtilis (B3) for postharvest control of peach brown rot. Plant Dis 72:622 626 Strobel G (2006) Muscodor albus and its biological promise. J Ind Microbiol Biotechnol 33:514 522 Strobel GA, Dirkse E, Sears J, Markworth C (2001) Volatile antimicrobials from Muscodor albus, a novel endophytic fungus. Microbiology 147:2943 2950 Takeda F, Krewer G, Andrews EL, Mullinix B, Peterson DL (2008) Assessment of the V45 blueberry harvester on rabbiteye blueberry and southern highbush blueberry pruned to v-shaped canopy. HortTechnol 18:4 19 Trias R, Baneras L, Bados E, Montesinos E (2008) Bioprotection of Golden Delicious apples and Iceberg lettuce against foodborne bacterial pathogens by lactic acid bacteria. Inter J Food Microbiol 123:50 60 Wilson CL, Pusey PL (1985) Potential for biological control of postharvest plant diseases. Plant Dis. 69:375 378 Xu XB, Tian SP (2008) Reducing oxidative stress in sweet cherry fruit by Pichia membranefaciens: a possible mode of action against Penicillium expansum. J Appl Microbiol 105:1170 1177