Caldwell 2009

THE USE OF VINEGAR VAPOR AND POST-HARVEST BIOLOGICAL

CONTROL TO REDUCE PATULIN IN APPLE CIDER

By

Lucius Caldwell

B. Sc. McGill University, 2005

A THESIS

Submitted in Partial Fulfillment of the

Requirements for the Degree of

Master of Science

(in Food Science and Human Nutrition)

The Graduate School

The University of Maine

August 2009

Advisory Committee:

Alfred Bushway, Professor of Food Science and Human Nutrition, Co-Advisor

Beth Calder, Extension Food Science Specialist, Research Professor of Food

Science and Human Nutrition, Co-Advisor

Renae Moran, Associate Professor of Pomology

Rodney Bushway, Professor of Food Science and Human Nutrition

Vivian Wu, Assistant Professor of Food Science and Human Nutrition

THE USE OF VINEGAR VAPOR AND POST-HARVEST BIOLOGICAL

CONTROL TO REDUCE PATULIN IN APPLE CIDER

By

Lucius Caldwell

Thesis Co-Advisors: Dr. Alfred Bushway & Dr. Beth Calder

An Abstract of the Thesis Presented in Partial Fulfillment of the Requirements for the

Degree of Master of Science (in Food Science and Human Nutrition)

August 2009

FDA recently set limits on concentration of patulin in apple cider at 50 parts per

billion (ppb). One method to reduce patulin in cider involves reducing patulin-producing

molds on apples destined for cider pressing. Fumigations with peracetic acid (PAA) and

with vinegars have shown to cure infestations of various molds. Treatment with

Pseudomonad biocontrol agents (BCA) has shown to protect against further infestation

by molds. However, fumigation is prohibitively expensive for many cider producers, and

there is not yet a sufficient process that both cures and protects against mold infestation.

The efficacy of white distilled vinegar fumigation in curing an infestation of

Penicillium expansum on apples using a home humidifier was assessed. Other treatments

evaluated were vinegar pretreatment in combination with BCA and vinegar fumigation

versus PAA fumigation.

Apples were treated with or without BCA, inoculated by dipping in a suspension

containing 10 P. expansum spores mL" , placed inside a sealed room, and vinegar or

PAA was vaporized using a humidifier. Next, apples were treated with or without BCA,

inoculated again or not, wounded with a sterile glass rod, and stored in plastic tubs, at

room temperature, under a laminar flow hood for 2-3 weeks. After incubation, apples

were visually inspected for mold infestation; wounds were sampled, and viable mold

spores enumerated, using 3M™ Quick Swabs and 3M™ YM Petrifilms.

Vinegar fumigation was effective at reducing endemic molds to below detectable

levels on 89% of apples and reducing the 3 Log 10 P. expansum inoculation to

undetectable levels on 89% of apples. Vinegar fumigation was found to significantly

improve the efficacy of BCA at protecting against infestation of P. expansum (p<0.001).

Vinegar fumigation was found to be significantly more effective than PAA fumigation

(pO.OOl).

This research provides another measure for cider producers developing HACCP

plans that address the reduction of patulin in their cider.

ACKNOWLEDGEMENTS

In preparing to research, as well as throughout all aspects of my program, my two

co-advisors were invaluable. Professor of Food Science and Human Nutrition Dr. Alfred

Bushway and Extension Food Science Specialist Dr. Beth Calder were always available,

always kind and patient, and offered unparalleled generosity with their time, in addition

to being solid references for fact-checking and guiding research parameters. In addition,

I'd like to thank the other members of my committee, Dr. Renae Moran, Dr. Rodney

Bushway, and Dr. Vivian Wu, for providing support and guidance throughout the project.

Others whose support was invaluable include Katherine Davis-Dentici, Dr. L.

Brian Perkins, Dr. David Lambert, Dr. Seanna Annis, Dr. Chuck Moody, and Dr.

Byungchul Kim. Finally, I must thank my inspiring wife, Julie Caldwell, for providing

constant support and guidance during my pursuit of this project.

in

TABLE OF CONTENTS

ACKNOWLEDGEMENTS iii

LIST OF TABLES viii

LIST OF FIGURES ix

LIST OF ABBREVIATIONS x

INTRODUCTION 1

APPLES IN MAINE 1

PATULIN 2

PATULIN-PRODUCING FUNGI 4

FUNGAL PATULIN PRODUCTION 6

DIETARY SOURCES OF PATULIN 9

CIDER PRODUCTION 10

CURRENT PATULIN CONTROL MEASURES 11

CULLING ROTTEN FRUIT/REMOVING ROTTING PORTIONS OF FRUIT 15

CHLORINE 16

POST-HARVEST FUNGICIDES 21

MCP 22

DPA 24

REFRIGERATION 25

CONTROLLED ATMOSPHERE STORAGE 26

PASTEURIZATION/CHEMICAL PRESERVATION 28

iv

ALTERNATIVE PATULIN CONTROL MEASURES 30

PATULNI REMOVAL MEASURES 30

PATULIN PREVENTION MEASURES 33

ELECTROLYZED OXIDIZING WATER 35

HEAT TREATMENT OF FRUIT 37

H202 38

ORGANIC ACIDS 40

VAPOR-PHASE ACID TREATMENTS 43

VAPOR-PHASE VINEGAR TREATMENTS 48

PERACETIC ACID 50

CHITOSAN 53

PLANT ESSENTIAL OILS 54

BIOFUMIGATION/MYCOFUMIGATION 57

BIOLOGICAL CONTROL 59

DEFINITION 59

HISTORY AND EMERGENCE OF MODERN CONCEPTS 60

PROCESS OF DEVELOPING BIOLOGICAL CONTROL AGENTS 63

PRE-HARVEST BIOLOGICAL CONTROL 65

POST-HARVEST BIOLOGICAL CONTROL 66

FUNGAL POST-HARVEST BIOLOGICAL CONTROL AGENTS 68

RHODOTORULA GLUTINIS 68

PICHIA SPP 69

CR YPTOCOCCUS SPP 70

v

METSCHNIKOWIA PULCHERRIMA 73

CANDIDA OLEOPHILA 76

CANDIDA SAITOANA 77

CANDIDA SAKE 78

AUREOBASIDIUM PULLULANS 79

BACTERIAL POST-HARVEST BIOLOGICAL CONTROL AGENTS 80

PANTOEA SPP 80

RAHNELLA AQAUTILIS 81

PSEUDOMONAS SPP 82

LACTOBACILLUS SPP 84

COMMERCIAL AVAILABILITY OF POST-HARVEST BIOLOGICAL

CONTROL AGENTS 85

EXPERIMENTAL OBJECTIVES 87

CURING 89

PROTECTING 91

OVERALL OBJECTIVES 92

MATERIALS AND METHODS 94

TRIAL ONE 100

TRIAL TWO 103

TRIAL THREE 106

STATISTICAL METHODS OF ANALYSIS 108

RESULTS I l l

TRIAL ONE 111

VI

TRIAL TWO 115

TRIAL THREE 119

DISCUSSION 126

SUITABILITY OF INOCULUM 126

EFFICACY OF THE BIOLOGICAL CONTROL AGENT 128

EFFECTS OF FUMIGATIONS 130

EFFECTS OF COMBINING FUMIGATIONS AND BIOCONTROL 136

CONCLUSIONS 142

FUTURE RESEARCH 145

REFERENCES 146

APPENDICES 179

APPENDIX A - PATULIN ANALYSIS 179

APPENDIX B - COUNT DATA 183

APPENDIX C - EFFECTS OF DATA TRANSFORMATIONS 187

BIOGRAPHY OF THE AUTHOR 190

vn

LIST OF TABLES

Table B.l. Data from Trial One 183

Table B.2. Data from Trial Two 185

Table B.3. Data from Trial Three 186

Table C.l. Effects of transformations on data from Trial One 187

Table C.2. Effects of transformations on data from Trial Two 188

Table C.3. Effects of transformations on data from Trial Three 189

vin

LIST OF FIGURES

Figure 1. Summary of treatments in Trial One 95

Figure 2. Summary of treatments in Trial Two 96

Figure 3. Summary of treatments in Trial Three 97

Figure 4. Yeast/Mold counts from un-inoculated apples in Trial One 111

Figure 5. Yeast/Mold counts from P. expansum-\nocu\atz& apples in Trial One 112

Figure 6. Yeast/Mold counts from un-inoculated apples in Trial Two 115

Figure 7. Yeast/Mold counts from P. expansion-inoculated apples in Trial Two 116

Figure 8. Yeast/Mold counts from un-inoculated apples in Trial Three 120

Figure 9. Yeast/Mold counts from P. expansum-mocvAaXed apples in Trial Three 121

Figure 10. Process flow diagram for apple cider production 144

Figure A. 1. MS/MS spectrum showing daughter scan of patulin standard 181

Figure A.2. Chromatogram of patulin standard (200 ppb), showing retention time and

daughter ions 182

Figure A.3. Chromatogram of sample, showing retention time and daughter ions 182

IX

LIST OF ABBREVIATIONS

Aw - Water Activity

BCA - Biological control agent

CFU - Colony forming units

GAP/cGAP - Current Good Agricultural Practice

GMP/cGMP - Current Good Manufacturing Practice

PAA - Peracetic acid

PDA - Potato-dextrose agar

PPB - Parts per billion

WV - White vinegar

x

INTRODUCTION

APPLES IN MAINE

The fruit of Malus x domestica trees, apples are a type of pome, a group of fruits

whose other members include cultivated varieties (cultivars) of pear and quince, as well

as wild varieties of hawthorn, loquat, brier, and medlar (Herausgegeben von A. Taufel

1979). Pomes are fleshy tree fruits whose seeds (pips) are embedded in a central cavity

within the core (Hessayon 1991).

Plants that produce pome fruit are hardy trees belonging to the subfamily

Maloidiae, within the rose family, Rosacea; the plants are variously called Pomoidiae or

Maloidiae. The apple is the most widely grown pome fruit in the world; it is cultivated or

grows wild in every temperate country, as well as in many Mediterranean and even

tropical countries (Blackburne-Maze 2003). Apples are not only the most popular pome

fruit, they are consistently among the top 3 most widely grown tree fruits in the U.S.

(Hessayon 1991; Moake and others 2005). Relics and remains of orchards indicate that

apples have been grown in the State of Maine since as early as the middle of the 17th

century, with the first commercial orchard being established in present day Orrington,

between 1804 and 1812 (Maine State Pomological Society 2009).

Over the last twenty years, apple cultivation has been declining within the State of

Maine. In 2002, although still the most important tree fruit crop in the State of Maine,

the apple was already losing ground; the number of acres in cultivation throughout Maine

had been halved since 1992 (Coverstone and others 2004). With cultivated acreage of

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apples declining and increasing pressures on the market coming from out of state and

overseas, many growers recognize cider production as a "loss-recovery mechanism",

whereby otherwise non-salable apples may be converted into a marketable product

(Rosenberger 2003; Koehler 2007).

Recent regulations are making the production and sale of cider an increasingly

greater challenge for small producers. U.S. FDA implemented 21 CFR §120 in 2001,

requiring cider producers to operate under Hazard Analysis Critical Control Point

(HACCP) plans. In 2005, U.S. FDA updated the Compliance Policy Guides Manual

published in August 2000, to represent, "the Agency's current thinking on its enforcement

process concerning the adulteration of apple juice, apple juice concentrates, and apple

juice products with patulin". This guide specifies a defect action level for patulin at 50

ppb, above which apple cider is considered to be adulterated with the toxin

(U.S.FDA/ORA2001).

PATULIN

Patulin is a fungal toxin or mycotoxin produced by over 60 species of fungi from

over 30 genera, including both commensal and pathogenic species found on the surface

of apples (Hasan 2000; Moake and others 2005; Kabak and others 2006; Murphy and

others 2006). Patulin (4-hydroxy-4H-furo[3,2c]pyran-2(6H)-one) is a low molecular-

weight lactone that is stable under the mildly acidic conditions found in apple juice and

cider, although it may degrade with exposure to sulfhydryl-containing compounds (Scott

and Somers 1968; Fliege and Metzler 2000; Sapers and others 2005).

2

Patulin was first isolated from Penicillium patulum (now called P. griseofulvum)

in the 1940s, and was extensively investigated as an antibiotic (Bennett and Klich 2003),

due to its inhibition of many fungi (Korzybski and others 1967) and at least 75 species of

gram positive and negative bacteria (Ciegler and others 1971).

Evidence suggests that patulin may be broadly toxic: there are reports of patulin

being mutagenic, neurotoxic, immunotoxic, genotoxic, and irritating to the

gastrointestinal tract of laboratory animals (Hopkins 1993). Results from a number of

studies in the early 1980s (McKinley and Carlton 1980a, b; McKinley and others 1982)

indicate that at least some of the toxicity of patulin (i.e. patulin-induced mycotoxicoses)

may be indirectly attributable to its antibiotic activity. Patulin was reported to irritate the

endothelia and mucosa of the gut, and to alter the ecological balance of gut microbiota,

thus bringing about ulcers, gastric distention, and in some cases, mortality.

A 2003 study (Liu and others) found patulin to be genotoxic, causing significant

oxidative damage to DNA in human-derived kidney cells and lymphocytes. A more

recent paper (Fuchs and others 2008) describes patulin-induced DNA damage to human-

derived liver cells. The authors report that damage caused by patulin is worse than

damage caused by several other known carcinogens, including polycyclic aromatic

hydrocarbons, nitrosamines, heterocyclic aromatic amines, and the mycotoxins

ochratoxin A and citrinin.

Although patulin is generally considered a low priority mycotoxin, with far less

toxicity than the aflatoxins or ochratoxin A, its presence may indicate that rotten apples

were used in the production of cider, which may pose other risks aside from patulin

adulteration (Moss 2002). Of particular concern is that different mycotoxins may interact

3

additively or synergistically (Speijers and Speijers 2004). Some research indicates that

different mycotoxins may exist within the same piece of fruit; patulin and citrinin have

simultaneously been isolated from Portuguese apples (Martins and others 2002), and at

least one microorganism (P. citrinum) produces both (Sweeney and Dobson 1998). An

individual who consumes a varied diet may ingest low levels of many mycotoxins, from

sources like grains, nuts, and juices; therefore, even though patulin itself appears to have

a low toxicity, it still is important to minimize exposure (Kumar and others 2008).

Any provisional allowance or defect action level must take into account the higher

levels of apple products that children consume, estimated to be as much as 6 times the

rate of adult consumption (Plunkett and others 1992). Many baby foods, juices, and other

products often consumed by children are sweetened with apple juice. Compounding the

issue, children and babies are generally more vulnerable to the effects of toxin-intake

than adults (Lewit and Baker 1995). These concerns were taken into consideration when

establishing the U.S. FDA 50 ppb defect action level and the CODEX Alimentarius 25

ppb limit on patulin concentration.

PATULIN-PRODUCING FUNGI

The most important and common patulin-producing fungi belong to three genera

of ascomycota: Penicillium, Aspergillus, and Byssochlamys (Bennett and Klich 2003;

Moake and others 2005). Penicillia include P. expansum, the common fungi responsible

for blue mold and brown rot in apples; P. crustosum; and P. roqueforti, the blue mold

used in production of many blue cheeses (Sommer and others 1974; Baert and others

4

2007). Interestingly, P. roquforti does not appear to produce patulin when growing on

cheese (Lopez-Diaz and others 1996). Naturally present in cheeses, sulfhydryl groups -

whose ability to accept hydrogen ions generated by oxidative ripening processes is

critical to the development of characteristic cheese flavors (Kristoffersen 1973) - bind

and degrade patulin, preventing toxin accumulation within cheese (O'Brien and others

2004). Aspergilli may also cause rot in apples, and include patulin producers such as A.

niger (common black mold) as well as A. giganteus and A. terreus (Kumpoun and others

2003). Byssochlamys species include the patulin producers B. fulva and B. nivea (Rice

and others 1977); the latter produces ascospores that may survive pasteurization

(Dombrink-Kurtzman and Engberg 2006).

While regional conditions and apple cultivar may determine locally relevant

molds, Penicillium expansum is the most important patulin producer associated with

apples (Bennett and Klich 2003). Most P. expansum strains assayed produce patulin

(Andersen and others 2004). P. expansum is a ubiquitous, soil-borne fungus (Domsch

and others 1980); therefore, many opportunities for infection of apples exist, from soil

and field bins, to packinghouse operations (Morales and others 2008b). P. expansum is

the most common storage rot of apples in many parts of the U.S. (Pierson and others

1971; Wilson and Nuovo 1973; Sholberg and others 2005b) and in Europe (Morales and

others 2008a).

5

FUNGAL PATULINPRODUCTION

Mycotoxin contamination of foodstuffs appears to be a unique health hazard,

which emerged after the development of techniques for storing crops. As apples are held

in storage, mycotoxin concentration generally rises, paralleling the growth of plant

pathogens and the development of storage rots. Fungal presence does not necessarily

equate to the production of toxins, but fungal growth and decay often do. While toxin-

producing fungi may be present on the surface of fruit, they will neither cause decay nor

produce toxins without some mode of entry into the flesh of the apple. Usually, fungi

gain access to the interior of fruit through a wound (Xu and Berrie 2005), although some

cultivars (such as Jonathon and Red Delicious) are susceptible to mycelial penetration

through the lenticels on fruit surfaces (Ogawa and others 1991). Over-mature or bruised

fruit is also particularly susceptible to fungal penetration through lenticel openings

(Wright and Smith 1954),.

Fungal intrusion and infection appear to be coordinated by volatiles (present at

high concentrations in the headspace above fruit during storage), which may act as a

signal, inducing fungal spores to germinate (Filonow 2001).

Numerous factors affect the microbial ecology of- and thus development of rot

on - fruit, such as geographic area, climate, pesticide and fertilizer applications

(especially the use of fungicides during the season and the application of a fertilizer or

amendment containing sufficient calcium), presence of antagonists on fruit and trees,

harvest method, post harvest fruit treatments, and storage conditions, (Doores 1983;

Sapers and others 2005). Storage humidity does not appear to have much effect on P.

6

expansion conidial germination, presumably because the wound site generally harbors

sufficient moisture (Ogawa and others 1991).

Apple cultivar seems to be a significant factor in whether fungi present will

colonize and decay fruit; variations in skin strength and thickness, as well as flesh

firmness, pH, sugar content, and concentration of antimicrobial compounds all affect

susceptibility to fungal infection (Sapers and others 2005). Open calyx apple cultivars,

such as Red Delicious, Jonagold, and Bramley, appear more susceptible to fungal

infections, which may go unnoticed and contribute to patulin contamination among these

cultivars (Moss 2002; Sapers and others 2005). Conversely, Golden Delicious apples

appear to be particularly resistant to accumulation of patulin, perhaps due to both

resistance to fungal infection and physiochemical character of the fruit flesh (Northold

and others 1978).

Fungal growth and patulin production are not always closely correlated. In some

cases, fungal growth is minimal, and patulin production is significant; in other cases,

fungal growth is extensive, but patulin production is negligible (Stott and Bullerman

1975b). Fungal patulin production is dependent upon numerous variables, including

substrate, water activity (Aw), temperature, pH, and nutrient availability (Acar and others

1998; Murphy and others 2006; Magan and Aldred 2007; Morales and others 2007a). In

a cider operation, patulin production may be determined by variability among the above

parameters in the fruit used to make juice, growth conditions throughout the season, fruit

maturity at harvest, extent of fungal contamination or rot development within fruit,

treatments of fruit and juice, and storage conditions of both fruit and juice (Sapers and

others 2005).

7

As a group, the mycotoxins are generally believed to be secondary metabolites -

byproducts of critical metabolic processes (Bennett and Klich 2003). If this is the case,

then searching for their function is moot: they are waste products. However, it is possible

that production of mycotoxins may confer some benefit to the fungi that produce them.

For instance, Penicillium verrucosum is one of many fungal species that produces the

mycotoxin citrinin; citrinin may protect the P. verrucosum from UV damage (Stormer

and others 1998)

Although the function of patulin is unknown, it may be ecological. As is the case

with many mycotoxins, patulin has antibiotic activity (McKinley and others 1982;

Boukouvalas and Maltais 1995). This may provide an evolutionary advantage, for

instance, by allowing the fungus to outcompete inhibited neighbors (Ciegler 1983).

Support for this theory may be provided by the evidence that patulin synthesis

appears to be a stress response; Grootwassink and Gaucher (1980) reported that patulin

production increases as growth rate declines. Hasan (2000) found that patulin production

increased during later phases of fungal growth, when nutrients are presumably beginning

to be depleted; Moake and others (2005) surmise nitrogen is the limiting nutrient. More

recent publications support the stress-induced toxin synthesis theory, and indicate that the

growth inhibitor and biocontrol enhancer 2-deoxy-D-glucose, as well as certain synthetic

fungicides, may actually increase patulin production by those molds that survive

treatment (Kazi and others 1997; Paterson 2007).

Interestingly, Penicillium expansum seems to inhibit some pathogenic bacteria

which may be present on fruit surfaces (Conway and others 2000). If P. expansum has

decayed an apple wound, Listeria monocytogenes was found to be unable to establish

8

itself and reproduce within that wound. Conway and others suggest that a reduction in

pH of the wound site may be a mode of inhibition, but patulin production was not

evaluated, and it is reasonable to suspect that the mycotoxin may also play a role in

inhibition, via antibiosis.

DIETARY SOURCES OF PATULIN

Foods that have been reported to contain patulin include wheat products (Lopez-

Diaz and Flannigan 1997), dairy products (Bullerman and Olivigni 1974), nuts (Jimenez

and others 1991), and fruit juices or foods that have been flavored with fruit juices

(Bennett and Klich 2003; Drusch and Ragab 2003).

Because they tend to harbor toxin-producing Penicillia molds, apple products

(such as cider and juice) are widely regarded as the most common source of patulin (Stott

and Bullerman 1975a; Thurm and others 1979; Bennett and Klich 2003; Murphy and

others 2006; Ricelli and others 2007). Patulin was first isolated from apples in 1956

(Brian and others 1956), and has since been isolated from apples and apple products in

every inhabited continent (Harwig and others 1973; Hasan 2000; Leggott and Shephard

2001; Martins and others 2002; Andersen and others 2004; Iha and Sabino 2008; Murillo-

Arbizu and others 2009).

Small cider producers who may utilize non-fancy apples, which would not be

suitable for eating, have been described as being more likely to produce cider that

contains patulin, due to the potentially lower quality fruit that may go into their product

(Brackett and Marth 1979). However, there is little evidence that this is actually the case,

9

and the multitude of variables involved in patulin production could just as easily mean

that juices produced at larger operations contain elevated levels of patulin.

It has long been known that patulin does not survive the fermentation process of

making hard cider, wine, or vinegar (Burroughs 1977; Westby and others 1997), as

Saccharomyces cerevisiae metabolizes the toxin into primarily E-ascladiol and Z-

ascladiol (Sapers and others 2005), as does the acetic acid bacteria Gluconobacter

oxydans (Ricelli and others 2007). Welke and others (2009) recently described a

cumulative reduction in patulin content through consecutive processing steps in

producing clarified apple juice, such as milling, filtering, enzymatic treatment and

pasteurizing; the combination of all steps yielded up to a 75% reduction in patulin

content.

CIDER PRODUCTION

Apple cider production in Maine is generally carried out by small producers who

grow their own apples. In most cases, growers produce cider from apples graded below

fancy, which are unsuitable for the fresh eating market; the State of Maine Department of

Agriculture Food Regulations for apple cider (Chapter 342) mandate that cider "shall be

pressed, squeezed or extracted from clean, sound apples that have been carefully

inspected and sorted to remove defects".

Growers usually store apples for at least a few days prior to pressing into cider,

either at ambient temperatures (deck storage), or under refrigeration or controlled

atmosphere (CA) storage. Controlled atmosphere storage is the holding of fruit at a

10

constant refrigerated temperature, while also controlling the composition of the storage

atmosphere, usually by reducing O2 concentration, increasing CO2 concentration, and/or

increasing N2 concentration. In some cases, growers will store apples for longer time

periods, either to press cider as needed or simply because more apples are being

harvested than can immediately be pressed into cider. Some growers sell apples to a

larger producer, in which case pre-processing storage times can range from a couple of

days to many weeks. Some processors press all fruit at once, and then freeze cider, while

others store apples and press cider as needed.

CURRENT PATULIN CONTROL MEASURES

Control steps for the reduction of patulin may be thought of as remedial or

preventive. Currently, no remedial measures to reduce patulin are widely practiced,

although certain unit operations involved in cider processing may reduce levels of patulin

present in juice, as mentioned above.

Measures that prevent the adulteration of cider with patulin focus on curing and/or

preventing the growth of patulin-producing molds (fruit surface sanitation), or inhibiting

the production of patulin by these molds. Discussed in depth below, these measures

include orchard phytosanitation (removal of diseased trees or portions of trees to reduce

pathogen inoculum density in the orchard), use of chlorine washes in fruit processing,

refrigerated storage of fruit and cider, controlled atmosphere storage of fruit, and the use

of synthetic fungicides on apples both in the field and post-harvest. While it is possible

to reduce or prevent patulin production by existing molds (e.g. by reducing storage

11

temperature sufficiently), the goal of a patulin management program should be to reduce

or eliminate these patulin-producing molds, ideally from the apple surface before fruit is

processed into cider. Pasteurization of cider can be thought of as a reduction of microbial

density, but it is by no means a complete sterilization of the juice; if heat resistant fungal

spores survive pasteurization (for instance, Byssochlamys nivea ascospores), and post­

processing patulin production thus continues at low levels, any removal, detoxification,

or adsorption of toxins would be moot without protection from further accumulation of

the toxin (Moake and others 2005). By addressing the problem of the toxin at the source

and dealing with molds that produce patulin rather than focusing on the toxic byproduct

of the molds, quality and other aspects of product safety will likely also improve. For

these reasons, fungal inhibition seems to be a more appropriate control measure than

removing or transforming patulin.

More than any other step, the most effective technique for reducing molds on

apple surfaces, and therefore reducing molds in cider, is the use of only sound, hand-

picked apples in cider production. While Penicillium expansum and other patulin-

producing fungi are often present on apple surfaces, the diseases these microorganisms

cause will not generally develop without a wound in the cuticle to allow fungal

penetration (Wilson and Nuovo 1973; Buchanan and others 1974; Sydenham and others

1995). Although the molds are certainly present on apple fruit, if no wound is present,

fungi will not be able to penetrate, multiply, and produce toxins within the apple, prior to

processing (Sanderson and Spotts 1995). Because wounds are the most common vector

of fungal penetration, any practice that reduces wounds will reduce both fungal decay and

patulin production within apples (Ogawa and others 1991; Jackson and others 2003).

12

Other measures to prevent adulteration of cider with patulin include adhering to

current Good Agricultural Practices (cGAP), such as proper tree care and orchard

phytosanitation, both during the growing season and in the off-season. cGAP are

recommended to improve airflow around trees and fruits, thus decreasing the likelihood

of fungal infestation, as well as to reduce pests that may damage fruit and allow patulin-

producing molds to colonize apples. Any pest that can break the skin of apples may be

considered a potential target in the reduction of patulin, since patulin-producing molds

cannot generally colonize the fruit without a wound or other means of ingress. Therefore,

control of insect, vertebrate, and other pests seemingly unrelated to patulin, may be

meaningful steps toward significant patulin reduction (Kabak and others 2006).

Other preventive measures include integrated pest management (IPM) and weed

control. By maintaining phytosanitary conditions, it is possible to reduce fungal load in

the orchard, decreasing the potential inoculum size of rot organisms, and also to reduce

mode of entry for fungal pathogens of fruit (Murphy and others 2006).

One effective method of inhibiting both fungal growth and patulin production is

to keep apples cold prior to pressing (Hasan 2000). Morales, Marin, and others (2007a)

found that, if apples were kept at 1 °C, rot organisms produced no patulin during 6 weeks

of storage, even if fruit showed visible damage from mold.

Post-harvest handling, such as the use of dip tanks and water flumes in the

processing of apples, has long been recognized as a source for the introduction or spread

of both human pathogens and storage diseases (including patulin-producing molds),

among produce (Beuchat 1996). The practice of using drench tanks to apply synthetic

fungicides and pre-storage anti-scald treatments may thus paradoxically increase the

13

incidence of Penicillia on stored fruit (Sanderson and Spotts 1995). If pathogens are

neither inactivated nor removed during washing, it is possible for them to infect the

majority of produce, rather than simply infecting sporadic pieces of fruit (Nguyen-The

and Carlin 1994; NACMCF 1999).

While sanitizers (primarily NaOCl) are commonly used to control the microbial

load of water used to process fruits and vegetables, it is a common misconception that

these sanitizers control microbial growth on the produce itself (NACMCF 1999). In

reality, much of the research evaluating the efficacy of sanitizers to reduce microbes on

the surface of produce has not reported sufficient control of pathogenic or rot microbes to

become widely accepted (Brackett 1999; Artes and others 2009; Olmez and Kretzschmar

2009).

Another point of concern when researching sanitizing agents or processes is that

some methods may control microbes on one type of produce quite well, but do not work

effectively for other fruits or vegetables. In other cases, an initial decrease in total

microbial load does not equate with decreased populations of important microbes after

storage. By reducing numbers of all microbes, it may be the case that handlers

inadvertently reduce numbers of antagonistic microbes that may otherwise control the

numbers of storage rot organisms or potential human pathogens during storage (Francis

and O'Beirne 2002).

14

CULLING ROTTEN FRUIT/REMOVING ROTTING PORTIONS OF FRUIT

Cutting out necrotic (rotten) portions of the fruit may reduce both patulin content

of apples and the population of molds on apples that produce patulin. Trimming P.

expansum decayed tissue from apple fruit has been found to remove between 93-99% of

patulin from the fruit (Lovett and others 1975). A review of 100 samples of cider by

Wilson and Nuovo (1973) reported that the highest levels of patulin were found in juice

from cider producers who did not sort out rotten apples. Sydenham, Vismer, and others

(Sydenham and others 1995) found that removing rotten and damaged fruit before

processing cider significantly reduced patulin content in the resultant juice. Hasan (2000)

measured patulin concentration in non-necrotic apple tissue at 66% of the level found in

flesh visibly decayed by P. expansum.

It appears that the patulin diffusion through fruit flesh is dependent upon fruit

characteristics such as firmness and density. While patulin penetrated the soft flesh of

tomato with ease, the toxin seems unable to diffuse through the firmer flesh of apples;

patulin concentration in apples has been found to drop off precipitously at a distance of

more than 2 cm from the zone of P. expansum infection (Rychlik and Schieberle 2001),

with only 2-6% of patulin migrating into surrounding tissue (Marin and others 2006).

More recently, Bandoh and others (2009) experimented with the removal of

patulin from apples by sequentially scooping decay out with a spoon, washing the wound

with water, and finally using a knife to trim sound fruit from around the decay. They

found that by scooping out decay with a spoon, they removed most patulin (98.3%);

washing wounds with water after removing decayed flesh further reduced patulin content

15

to trace levels throughout the remaining fruit; removing decayed flesh, washing, and

excising sound flesh immediately adjacent to the wounds, reduced patulin levels in the

remaining apples to undetectable levels.

As effective as culling may be, it is possible that internal infestations of patulin-

producing molds may go unnoticed, particularly in open calyx cultivars (Baldwin,

Braeburn, Delicious, Fuji, Jonagold, Gala, Golden Delicious, Munson, Rome Beauty,

Scarlet Pippen, and others). Labor costs also tend to be high for this type of unit

operation, and it may not be practical in larger operations. It seems prudent to

recommend that culling rotten fruit and rotten tissue should be an important practice, but

by no means the entire control plan to reduce patulin in a juice production operation.

CHLORINE

Chlorine, most commonly used in the form of NaOCl, is utilized in cider

processing to prevent the growth of microorganisms suspended in wash water, thereby

maintaining sanitation of the water used throughout numerous unit operations. Hasan

(2000) found that a 5-minute dip in a 3% NaOCl solution (full-strength bleach) cured

infestations of Penicillium expansum and other storage rots on apple surfaces, and

completely prevented toxin formation by numerous fungal species. However, dipping

fruit in full-strength bleach may damage fruit or impart fruit with a bleach odor, and is a

potentially very expensive precaution.

Others have examined the potential antifungal activity of dilute solutions of

chlorine (CI), using CI concentrations similar to those recommended by state and federal

16

agencies for controlling microbes in wash water. Chen and others (2004) found that a 5-

minute wash in a solution of NaOCl (providing 200 ppm CI) delays but does not prevent

the growth of P. expansum on apples. Spotts and Peters (1980) found that dipping pears

in a dilute solution of NaOCl reduced populations of P. expansum on fruit. While CI

washes may help reduce the patulin content of juice, most research has unfortunately

revealed that, when appropriately diluted, CI is not capable of reducing populations of P.

expansum by more than 2 logio.

Longer duration (7 min) dips, in solutions with higher than recommended

concentrations of chlorine (800 ppm available CI), followed by brushing and rinsing, has

been reported to substantially reduce the fungal causative agents of fly speck and sooty

blotch (Batzer and others 2002). This type of treatment would likely decrease

populations of patulin-producing molds, and would remove rotting or necrotic tissue that

could potentially contain significant amounts of pre-formed patulin. However, this series

of steps is very labor intensive, and the level of chlorine used is not ideal for numerous

reasons, as discussed below.

There is substantial evidence that chlorine reduces bacterial pathogens on

produce. At 100-200 ppm, chlorine has been reported to significantly reduce Listeria

monocytogenes on lettuce (Beuchat and others 2004), and Salmonella spp. and

Escherichia coli on various fruits and vegetables (Zhuang and others 1995; Sapers and

others 1999; Weissinger and others 2000).

There are many environmental and health issues associated with chlorine use,

such as the formation of toxic byproducts, including chloroform, carcinogenic

trihalomethanes, haloacetic acids, and chloramines (Suslow 1997; Nieuwenhuijsen and

17

others 2000; Artes and others 2009). As a result, Germany, The Netherlands, Denmark,

Switzerland and Belgium have chosen to prohibit the use of chlorine in the production of

fresh-cut produce (Betts and Everis 2005).

Chlorine dioxide (CIO2), a stable, water-soluble gas, is a strong oxidizer that has

been described as an effective antimicrobial agent. CIO2 inhibits bacterial endospores,

planktonic and sessile bacteria, fungi, and viruses, and does not react with organic

molecules to produce carcinogenic byproducts (Artes and others 2009). CIO2 is an

effective sanitizer for use on equipment (Kreske and others 2006).

As of the time of writing, there are no reports of CIO2 being used to inhibit P.

expansum, specifically. Neither are there any reports of CIO2 being use to control fungal

rots in apples. However, there is a wealth of literature detailing the use of CIO2 on other

produce. Treatment of produce with CIO2 has been found to provide a 5 log 10 reduction

of certain microorganisms. The technology appears promising, and CIO2 may present an

option for growers looking to reduce populations of patulin-producing molds, human

pathogens, storage rots, and cider spoilage organisms on the surface of apples.

Immersing broccoli sprouts for 5 minutes in a solution of 50 ppm CIO2 was

shown (Kim and others 2009) to reduce pre-existing populations of yeast and mold by

approximately 1.5 logio; although modest, this microbial reduction was found to persist

over a 6 day storage period. When the same researchers combined 50 ppm CIO2 with

0.5% fumaric acid, they observed an improved efficacy to an approximately 2.5 logio

reduction of yeast and molds, which again persisted through storage. The authors

speculate that the improvement in efficacy may have been due to solution pH

optimization to maintain the maximum available chlorine.

18

Treatment with 50 ppm gaseous CIO2 has been reported (Jin-hua and others 2007)

to reduce the incidence of fungal rot in green bell peppers. After 30 days, none of the

treated fruit had developed any visible fungal rot; after 40 days, only 9% of the treated

peppers had developed visible rot, compared to visible rot in almost 40% of the untreated

peppers.

Treatment with 15 ppm gaseous CIO2 has been reported to reduce yeast and mold

populations on blueberries, as well. Wu and Kim (2007) report that, while a 10 second

immersion provided only a 0.6 logio reduction, a 1-minute immersion effected a 1.8 logio

reduction in the population of yeasts and molds on blueberries. With longer immersion

times of 1-2 hours, observations of 2.5-3 logio reductions in yeast and mold populations

were reported, even when using only 10 ppm CIO2.

Others (Gomez-Lopez and others 2007) have reported a 0.7 logio reduction of

yeasts and molds on grated carrots treated with gaseous CIO2 for 6 minutes, during which

time the CIO2 reached a maximum concentration of 1.33 mg/1.

CIO2 appears to be a broadly effective antimicrobial agent, and CIO2 treatment

may inhibit bacterial plant pathogens and storage rots. Erwinia amylovora is the bacterial

causative agent of fire blight, a major disease of apples and pears (Winslow and others

1920). Fire blight may be thought of as both a plant pathogen that affects live trees in the

orchard, as well as a spoilage organism that reduces quality of stored fruit by causing

"bacterial ooze" (Wilcox 1994). In trials involving plant pathology, CIO2 was found to

be ineffective at eradicating E. amylovora from apple fruit (Roberts and Reymond 1989).

The apple cider spoilage organism Alicyclobacillus acidoterrestris is a

thermophilic spore-former, common in apple orchards, and an occasional spoiler of

19

acidic juices. Using commercially available ClC>2-generating sachets to generate low,

medium, or high release gaseous CIO2, Lee and others (2006) treated A. acidoterrestris-

inoculated apples with CIO2 for 30 minutes, 1, 2, or 3 hours. When the low-release

sachet was used (maximum CIO2 concentration of 0.4 mg/L), one hour of exposure to

CIO2 effected a 2.7 logio reduction, and three hours of exposure to CIO2 effected a 4.5

logio reduction, in the number of A. acidoterrestris on apple surfaces, without any

reduction of visual quality in the apples. When using the medium release sachet

(maximum CIO2 concentration of 0.6 mg/L), or the high release sachet (maximum CIO2

concentration of 0.8 mg/L), one hour of exposure to CIO2 was enough to reduce spores of

A. acidoterrestris to undetectable levels (a 5 logio reduction). However, exposure to

these higher concentrations of CIO2 was found to damage the apples and reduce quality.

Evidence suggests that CIO2 may inhibit a great diversity of microorganisms on

produce, including bacterial human pathogens, adding incentive for growers and

producers to utilize this emerging technology. Reductions of the human pathogens E.

coli 0157:H7, L. monocytogenes, and Salmonella typhimurium have been reported for

broccoli sprouts treated with CIO2 (Kim and others 2009). Others have reported up to 5

logio reductions of L. monocytogenes (Du and others 2002) and greater than 5 logio

reductions of E. coli 0157:H7 (Du and others 2003) on apple surfaces, following

treatments with gaseous CIO2.

Dipping produce (apples, lettuce, strawberries, and cantaloupe) for 5 minutes in a

solution of 3-5 ppm CIO2 has been shown to reduce numbers of the human pathogens E.

coli and L. monocytogenes by 5-5.6 logio. However, when the produce was then stored at

20

4 °C for 9 days, this treatment reportedly led to an increase in yeast and molds on the

produce (Rodgers and others 2004).

Chlorine may not be appropriate for reducing the fungal load on apple surfaces,

but it may reduce populations of potentially pathogenic bacteria on fruit surface. Also, in

operations using flume water or a dunking wash step, chlorine remains an important

chemical treatment for reducing cross-contamination.

POST-HARVEST FUNGICIDES

Currently, a limited number of fungicides are EPA registered for post-harvest use

on pome fruits; thiabendazole used to be, and captan now is, the most popular synthetic

fungicide used post-harvest on apples, though others are available (Rawn and others

2008). Synthetic fungicides may reduce populations of extant molds, and protect against

future infestations of molds, including those that cause patulin, but their efficacy is being

eroded by overuse and subsequent resistance.

Thiabendazole, imazalil, and sodium ortho-phenylphenate were, for a number of

years, the most popular post-harvest fungicides used on fruit (Amiri and others 2008).

For over twenty years, there have been reports of resistance to thiabendazole, imazalil,

and benamyl among Penicillium and Botrytis species across the U.S. and Europe (Leroux

and Clerjeau 1985; Bus and others 1991; Chand-Goyal and Spotts 1997; Errampalli and

others 2006).

More recently, the fungicides pyrimethanil and fludioxonil have been introduced,

yet pyrimethanil resistance has long been observed (Chapeland and others 1999).

21

Although fungal resistance to fludioxonil has not been observed in the field yet, mutants

that possess some degree of resistance have been observed in laboratory cultures (Faretra

and Pollastro 1993; Ziogas and Kalamarakis 2001).

In a comprehensive study by Bryk (1991), nine common fungicides were tested

for their ability to protect fruit from infection with P. expansum and two other post-

harvest storage pathogens of apples (Botrytis cinerea and Monilinia fructigena). Apples

were dipped in thiophanate-methyl, bitertanol, flusilazol, pyrifenox, procymidone,

captan, dithianon, iminoctadine DBS, or iminoctadine, then inoculated with 1 of 3 fungal

pathogens, and stored for 3 days at 20 °C. Of the three fungal pathogens tested, P.

expansum was controlled the least effectively by all nine fungicides. Flusilazol and

procymidone provided the most effective control of P. expansum-inductd decay,

preventing blue mold rot in only 43.6 and 41.6% of fruit, respectively. Less than 50%

control is unacceptable, especially considering the costs and concerns associated with

post-harvest applications of synthetic fungicides.

MCP

1-Methylcyclopropene (MCP) is a synthetic ethylene action inhibitor, which

delays ripening in apples and other fruit by inhibiting enzymes involved in the synthesis

of ethylene and other plant volatiles (Fan and Mattheis 1999). Apples may be exposed to

a post-harvest gas-phase MCP treatment, which slows maturation of fruit. The efficacy

of MCP is affected by cultivar and appears to work best under controlled atmosphere

storage (Watkins and others 2000).

22

While MCP has not been extensively researched for inhibitory activity against P.

expansum, delayed ripening of fruit is thought to delay or inhibit storage pathogens, such

as P. expansum, since less-ripe fruit is considered more resistant to infection (Baritelle

and others 2001). In general, post-harvest disease resistance of produce is closely linked

to the ripening process, with resistance dropping markedly at the onset of tissue

senescence and decay (Mari and Guizzardi 1998).

In addition to delaying ripening, there is evidence that MCP may inhibit storage

pathogens directly, by impeding pathogen-host identification associated with elevated

levels of ethylene such as those found regularly in apple storage areas. Environmental

ethylene induces conidial germination of P. expansum spores in water (Kepczynski and

Kepczynska 1977) and on apple surfaces (Eckert and others 1992). This stimulation of

reproduction has been similarly described in other Penicillia, and it is thought that

ethylene-induced germination may be a potential mechanism of host identification by

fungal pathogens (Droby and others 2008). If ethylene production is inhibited, it is likely

to presume that fungal germination will decrease, thereby inhibiting growth of fungal

storage pathogens, such as P. expansum.

However, this is not the case with strawberries, and evidence (Janisiewicz and

others 2003; Leverentz and others 2003) suggests that, in certain instances, treatment

with MCP may increase populations of P. expansum on apples. The authors speculate

that the mode of action involves MCP reducing or inhibiting activity of key enzymes

associated with plant defense responses. Effects were mitigated by combining MCP,

which is more often used to improve storage quality than reduce storage rots, with other

post-harvest treatments specifically intended to inhibit storage pathogens. Others

23

confirm this idea and recommend that MCP be included in an integrated decay control

strategy, but not be relied on as the sole measure of decay control (Spotts and others

2007).

DPA

Diphenylamine (DPA) is a synthetic, volatile antioxidant used in making

explosives, propellants, perfume, and rubber (Drzyzga 2003), that has also been used for

decades on storage apples to prevent the development of scald. DPA inhibits the

oxidative degradation of plant volatiles during storage that ordinarily lead to the

accumulation of phytopathogenic chemicals in the skin of apples, causing scald

(Bramlage 1988; Whitaker 2000).

While DPA is not widely used to control rot organisms such as Penicillium

expansum, its use as a scald inhibitor is very popular, and it may help control secondary

infections of fruit. Work by Spotts and others (1988) suggests that, in some cases, DPA

may actually increase the incidence of blue mold rot when P. expansum is present, by

facilitating fungal penetration into core fruit tissue. Nonetheless, DPA appears to prevent

scald disease from damaging apple skin sufficiently enough to allow the opportunistic

pathogen to colonize fruit and cause blue mold. Evidence also suggests that some strains

of P. expansum are sensitive to DPA treatment, especially those strains that are resistant

to benzimadole fungicides (Rosenberger and Meyer 1985; Rosenberger and others 1991).

Consequently, a combination of benzimadole and DPA was used successfully for a

number of years to control P. expansum among stored apples, with the thought being that

24

benzimadole would inhibit fungicide-sensitive strains of P. expansum, and DP A would

inhibit those benzimadole-resistant strains of P. expansum.

However, this treatment regime failed to account for strains with moderate

benzimadole-resistance, which were not controlled by either the benzimadole or DPA

(Rosenberger and others 1991). More recent work (Sholberg and others 2005a) indicates

that DPA-sensitivity may be waning among P. expansum, with more strains resistant to

the effects of the scald inhibitor.

Additionally, there are concerns over persistence of DPA in the environment

(Drzyzga 2003) and potential toxicity to bacteria and animals (Flaim and Toller 1989).

One group (Murin and others 1997) has written that DPA should be considered of high

risk for the aquatic environment. This hazard may be in part due to the fact that, in the

presence of UV-light, DPA readily undergoes photocyclization to carbazole (Sur and

others 2000), a structural analog of dibenzo dioxins (Kronberg 2008). Carbazoles have

been known to bio-accumulate (Southworth and others 1979), are highly toxic to aquatic

organisms, and may cause long-term environmental damage (Drzyzga 2003).

REFRIGERATION

If starting with sound apples, one highly effective method of inhibiting both

fungal growth and patulin production is to keep apples cold prior to pressing, and to

immediately chill pressed juice (Hasan 2000; Morales and others 2007a). P. expansum

growth and ability to cause decay on apples is effected by temperature: maximum growth

rate of the fungus is near 20 °C; growth rate drops off quickly and precipitously as

25

temperature is reduced to 0-10 °C; and growth rate tapers off more shallowly and

asymptotically as temperature is increased to 30-40 °C (Khan and others 1993).

In stored apples, patulin production also appears to reach a maximum near 20 °C

(Paster and others 1995; McCallum and others 2002). Current recommendations call for

storage of apples and juice at 41 °F (5 °C), but the recent work of Baert and others (2007)

indicates that 5 °C may not be low enough to prevent toxin formation. These researchers

found that, although fungal growth was retarded, patulin production relative to mycelial

mass increased as temperature was reduced from 20 °C to either 10 °C or 4 °C; patulin

production decreased only when temperatures were between 4 °C and 1 °C. These results

indicate that even at common refrigeration temperatures, appreciable amounts of patulin

may be produced. Only by storing apples and juice at temperatures near 0 °C can patulin

production be inhibited with refrigeration. These findings are supported by work of

Morales, Sanchis, and others (2007b), who found that apples stored at 1 °C did not

accumulate patulin during 6 weeks of storage, even if visibly damaged by mold.

CONTROLLED ATMOSPHERE STORAGE

Some producers store apples, and then press cider continually throughout the

year. Although it is possible to store refrigerated apples ambient air for shorter durations

(up to approximately 4 months), controlling the atmosphere of the storage area offers

many advantages. After initial processing of apples (such as harvesting, sorting,

washing, grading), storing fruit in a I0W-O2, high-C02 controlled atmosphere (CA) before

pressing into cider may prevent molds from growing, reproducing, and/or spreading

26

among the stored apples (Morales and others 2007b). Fungal rot organisms tend to grow

well at the O2 concentration of ambient air (21%), but are inhibited as O2 concentration

approaches 2%. As O2 concentration decreases from 2% to 1 %, fungal growth rate drops

by 50% (El-Goorani and Sommer 1981). Low O2 conditions would likely inhibit patulin

synthesis as well as fungal growth, since fungal hydroxylases and dehydrogenases

involved in patulin biosynthesis have been found to require both O2 and NADPH to

function (Moake and others 2005).

L0W-O2 and/or high-CC^ controlled storage atmospheres have also been reported

(Kader 1995) to delay ripening of climacteric fruit (such as apples), and to reduce both

rate of respiration and rate of ethylene production. Kader (1995) found that CA

conditions inhibit ethylene synthesis enzymes (ACC synthase and ACC oxidase), reduce

the internal pH of fruit, lead to the accumulation of antimicrobial compounds within

flesh and skin, and manipulate both redox reactions and the electron transport system in

fruit tissues. These effects may stimulate plant defense responses or otherwise inhibit the

growth of rot fungi on the surface of fruit. CA may also delay the degradation of

nutritional content and the decline of fruit quality (as measured by indices such as color,

firmness, and flavor) that normally occur during storage.

However, Sholberg and others (2005b) report that the incidence of fungal rot in

fruit stored under CA has recently been increasing. In some cases, the incidence of rot

among fruit stored in CA has exceeded that found in fruit stored in ambient air. The

authors suspect that insufficient cleaning and sanitization of CA storage facilities may

have allowed or even selected for the survival and persistence of low-oxygen tolerant

isolates of some rot fungi. These findings demonstrate the importance of thorough

27

sanitation of all equipment, so as to avoid cross-contamination of the crop from year to

year. Additionally, CA does not cure extant molds that have already infested the surface

of apples, but simply controls the growth and spread of fungi among stored fruit.

PASTEURIZATION/CHEMICAL PRESERVATION

After pressing the apples into cider, pasteurizing and adding chemical

preservatives to the juice may reduce populations of patulin-producing molds, and

chemical preservatives may help to prevent molds from reproducing (Lennox and

McElroy 1984; Roland and Beuchat 1984). This is the most well known and well used

example of how effective control of microorganisms must combine a pathogen reduction

technique (in this case, pasteurization) with a protective technique (in this case, addition

of a chemical preservative). Pasteurization and chemical preservatives provide a fair

margin of safety and protection; the combination of both steps enhances the respective

capability of each technique.

Though not commonly found, the patulin-producing fungus Byssochlamys nivea

has been shown to produce ascospores that can survive the pasteurization process, as

previously mentioned (Dombrink-Kurtzman and Engberg 2006). Also, while the popular

preservatives propionate and sorbate may at one time have effectively prevented mold

growth and/or patulin production (Lennox and McElroy 1984; Ryu and Holt 1993), their

effectiveness may be declining. Suhr and Nielsen (2004) have written that propionate

does not inhibit most spoilage fungi tested, stimulates the growth of some Penicillium

species (especially at high Aw levels, such as those found in juice), and may increase

28

toxin production in certain strains. Chen and others (2004) found that sorbate inhibited

neither the growth of P. expansum, nor patulin production.

Non-thermal methods of pasteurization using ozone (Choi and Nielsen 2005; Wu

Xiao and others 2006), irradiation (Aziz and Moussa 2002), and pulsed electric fields

(Evrendilek and others 2008), have been found to effectively inactivate P. expansum in

apple and other fruits, as well as juices and nectars. Processing juice with ozone may

decrease quality (Choi and Nielsen 2005), and all three techniques may degrade

anthocyanins and other antioxidants in juice (Tiwari and others 2009). These

technologies may be promising, but as of yet, have not been extensively tested.

Resistance of many producers and growers to their use also presents an obstacle to

widespread acceptance.

The above described techniques currently provide a modicum of efficacy for

reducing patulin in apples and apple products; at this point, these may be sufficient for

the needs of many growers and cider producers. The needs of organic growers and cider

producers may not be currently met, as regulations restrict the use of many synthetic

chemicals (including fungicides). However, organic operations are not alone in requiring

a better means of reducing molds that both produce patulin in cider and degrade the

quality of stored apples. As synthetic fungicides (the only current option available for

growers looking to simultaneously cure and protect against post-harvest infections of

patulin-producing fungi) lose efficacy, treatment costs continue to rise, and concerns

mount regarding environment and worker health, scientists must research sustainable

alternatives to synthetic fungicides for fruit surface phytosanitation. To avoid fungicides,

growers and producers will likely need to combine separate curative and protective steps.

29

AL TERNA TIVE PA TULIN CONTROL MEASURES

Numerous techniques to reduce patulin in juice or cider have been explored.

Again, they may be generally categorized as either remedial measures (techniques meant

to reduce or remove patulin from juice after the toxin has been formed) or preventive

measures (techniques meant to inhibit the production of patulin by capable fungi).

PATULIN REMOVAL MEASURES

Remedial measures to remove patulin after it has formed include filtering juice

through charcoal or through other media (Sands and others 1976; Leggott and others

2001; Kadakal and Nas 2002), introducing ozone into juice (McKenzie and others 1997),

irradiating juice (Yun and others 2008), using bacterial biotransformation of patulin into

byproducts with reduced toxicity (Ricelli and others 2007), and using chemicals such as

cysteine (Morgavi and others 2003), sulfur dioxide, or ascorbic acid (Aytac and Acar

1994; Carlas and Aranda 1996; Drusch and others 2007) to chelate or detoxify patulin.

While patulin is quite stable in an acidic environment, such as apple cider, there

are some reports that patulin is reduced by treating juice with heat, such as by

pasteurizing (Kadakal and Nas 2003). According to Murphy and others (2006),

variability among reported effects of heat on the degradation of patulin in juice are the

result of variability among acidity of the juices tested. The authors deduce that

researchers who investigate disappearance of patulin from low acid juices tend to report

30

greater reductions than those who investigate disappearance of patulin from more acidic

juices, in which patulin is more stable. This claim is backed up by McCallum and others

(2002), who report that patulin is unstable at high pH.

Filtration is currently a unit operation in the production of apple juice, but not

cider. The presence of apple pulp and retained solids are what distinguish cider from

juice, and filtration techniques reduce pulp and solids. Filtration may be appropriate in

apple juice production, where clarity and stability are valued more highly than retention

of solids, flavors compounds, and pulp that are integral to apple cider.

Although removing patulin after its formation may be possible, it does not seem

to be optimal. Patulin has been described as a reliable indicator of the quality of fruit

used in juice production (Gaspar and Lucena 2009), and it is reasonable to suspect that if

patulin was present at one time, other toxins or compounds that may compromise quality

or safety of the final product are likely to persist. Additionally, juice that previously

supported mold growth may contain as-yet undiscovered toxic compounds, such as

breakdown products of fungal metabolites or even allergenic or inflammatory cellular

structures.

Similar arguments counter the potential for detoxification or decontamination of

patulin by microbial digestion of the toxin into smaller precursors or metabolites.

Gluconobacter oxydans has been reported to convert patulin into a mixture of the E- and

Z- isomers of ascladiol (in a 3.5:1 ratio, respectively) (Ricelli and others 2007).

Ascladiol appears to have one fourth the acute toxicity of patulin, but chronic toxicity of

ascladiol has not yet been studied.

31

Saccharomyces cerevisiae and Pichia ohmeri have been found to significantly

degrade patulin in apple juice, although metabolites were not identified (Coelho and

others 2008). Moss and Long (2002) report that S. cerevisiae also degrades patulin

(primarily to E-ascladiol, with smaller amounts of Z-ascladiol being generated as well),

although the yeast only does so under anaerobic conditions.

E-ascladiol, the main degradation product of these biotransformations, is simply

dehydroxylated patulin. In the patulin biosynthesis pathway of most toxin-producing

fungi, E-ascladiol is oxidized to patulin without any intermediate steps (Moake and

others 2005). Stability of ascladiol has not been fully assessed, and it seems likely that

reversion of ascladiol to patulin is possible, especially if live fungi are present.

Lactic acid bacteria from the genera Lactobacillus and Bifidobacteria have been

reported to potentially metabolize patulin, and may also adsorb the toxin onto their

surface (Fuchs and others 2008). These microbes may thus provide a means to both

detoxify and selectively remove patulin, but their full potential has not yet been realized,

partially because LAB may spoil juice (Shearer and others 2002).

Detoxification or decontamination of juice that contains patulin may be possible,

but should follow certain guidelines. Kabak and others (2006) report that FAO has

adopted a set of guidelines (Van Egmond and Jonker 2004) for the decontamination or

detoxification of mycotoxins in foods, based on work published in the Journal of the

AOAC by Park and others (1988). The article of Park and others (1988) concerned

ammoniating grains and meals to detoxify aflatoxins. The authors maintained that

successful decontamination of foods requires destruction, inactivation, or removal of the

toxin; avoidance of producing or leaving toxic residue; maintenance of desirable physical

32

and sensory properties of the food; destruction of fungal spores and mycelium; and

technical and economical feasibility. As of today, there is not a technique available that

meets all of these needs. Mycotoxin decontamination may be a valuable part of a

strategy to reduce patulin in cider, but it could never be the sole method in a patulin

reduction plan.

PATULIN PREVENTION MEASURES

Because patulin removal is difficult in foods, prevention of the growth of molds

that may produce patulin seems the best approach for reducing levels of patulin in cider.

Patulin appears to accumulate within apples primarily post-harvest; nonetheless, pre-

harvest treatment of fruit and trees may affect susceptibility to fungal infection and

subsequent patulin production (Sapers and others 2005). Numerous pre-harvest

techniques may be employed by apple growers to reduce molds on the surface of apples,

by reducing pests, diseases, or conditions that may lead to mold infestation. These are all

important methods, not only for the control of patulin, but for quality assurance, crop

disease management, and control of pathogens. However, without proper handling of the

apples post-harvest, all work done pre-harvest will be for naught.

Assuming adherence to cGAP, healthy trees, and sound apples, post-harvest

methods to reduce patulin should focus on reducing populations of endemic molds and

protecting against further fungal infestation. This concept may be neatly described as

fruit surface sanitation.

33

Unfortunately, the only commonly available technique for simultaneously curing

and protecting against further infestations of patulin-producing fungi involves

applications of synthetic fungicides that have at least some degree of persistence.

Synthetic fungicides certainly may be an effective part of disease management in the

orchards, when they make up a portion of integrated pest management. However, post-

harvest application of fungicides has drawbacks, as previously mentioned, the greatest of

which is the declining efficacy of these chemicals. Furthermore, there is a growing

segment of growers and producers who refuse to use synthetic fungicides during the

season or post-harvest.

When discussing alternative treatments, it is important to keep in mind that the

attributes of any technique most relevant to growers and producers are cost, efficacy, and

simplicity. Also, if any single method can solve numerous problems (for example, one

treatment controlling toxin-producing molds, foodborne pathogens, and spoilage

organisms), this technique would be highly useful for apple growers and/or cider

producers.

Luckily, there is a wealth of research into alternative treatments that are effective

at curing or protecting against mold infestations. Unfortunately, these newly described

techniques do not yet adequately, reasonably, and accessibly offer both curative and

protective capability.

34

Electrolyzed oxidizing water

Electrolyzed, or Electrolyzed oxidizing, water (EO H2O) is basically a specialized

chlorine treatment, first described in Japan in the early 1990s (Shimizu and Hurusawa

1992). By electrolyzing a H20-based solution of NaCl, it is possible to provide

antimicrobially significant quantities of available CI from ordinary table salt (Izumi

1999).

The main advantages of EO H2O include effective disinfection, ease of operation,

low operating costs, and a reduced environmental impact. The drawbacks include

potentially high start-up and equipment costs, as well as the rapid loss of antimicrobial

activity that follows cessation of electrolysis (Huang and others 2008).

EO H2O may help prevent cross contamination of fruit during washing steps.

Okull and LaBorde (2004) report that a 1-minute immersion in a solution of EO H2O (33

to 60 ppm available CI) did not reduce the incidence of decay in wound-inoculated

apples, but did reduce Penicillium expansum spores in the wash water by nearly 5 logio.

While no studies to date have reported the reduction of P. expansum or other

fungal pathogens on the surface of apples, there are numerous inplanta studies

confirming the efficacy of EO H2O at controlling pathogens on other produce.

EO H2O may be able to provide control of fungi, including storage rots. Briefly

immersing strawberries and cucumbers into an EO H2O solution (30 ppm available CI)

reduced latent populations of fungi by 1.5-2 logio (Koseki and others 2004). Others (Al-

Haq and others 2001) report a reduction, but not complete control of, brown rot caused

by Monilinia fructicola in peaches. The researchers report that 5 minutes of immersion

35

into a solution of EO H2O (290 ppm available CI), delayed the onset of brown rot in non-

wounded fruit that was spray-inoculated with a suspension of M. fructicola spores (5 x

105 CFU mL"1). The EO H2O treatment did not control rot in fruit that had been wounded

prior to inoculation, highlighting the importance of handling and care of fruit to avoid

damage. In a later paper, the same authors (Al-Haq and others 2002) studied fungal rot

of pear; a 10 minute immersion into a solution of EO H2O (200 ppm available CI)

controlled Botryosphaeria berengeriana (formerly B. dothidea), the fungal causative

agent of canker in pear.

From information available, it appears that fungi are more resistant than bacteria

to the effects of chlorine. Nonetheless, the antibacterial activity of EO H20 is a benefit

that makes it very attractive to many growers or producers of agricultural products, and

numerous studies have confirmed in vitro antimicrobial activity of EO H2O against gram-

negative and gram-positive bacteria. In a review, Huang and others (2008) report that

treating produce with EO H2O (<85 ppm available CI) provided complete control of the

human pathogens Escherichia coli 0157:H7, Salmonella enteriditis, S. typhimurium,

Listeria monocytogenes, Staphylococcus aureus, and Enterobacter aerogenes.

Izumi (1999) reported that immersing produce for 3 minutes in a solution of EO

H2O (50 ppm available chlorine) reduced by 2.6 logio the number of mesophilic aerobes

on carrots, spinach, peppers, potatoes, and radishes. Park and others (2001) reported

nearly identical reductions of E. coli and L. monocytogenes populations, after immersing

lettuce for 3 minutes into an EO H2O solution (45 ppm available CI).

36

Heat treatment of fruit

First written about for use with apples in 1978 (Liu), and then studied on many

crops by Klein and Lurie (1992), heating fruit after harvest to reduce post-harvest

pathogens has been reported extensively by Leverentz (Leverentz and others 2000;

Leverentz and others 2003). The earlier groups (Liu 1978; Klein and Lurie 1992) found

that heat treatment was highly effective at both maintaining indices of quality in stored

apples and reducing or eliminating fungal storage rots. The Leverentz group (Leverentz

and others 2000; Leverentz and others 2003) has found that if Gala or Golden Delicious

apples were held at 38 °C for up to 4 days, the heat treatment had a curative effect on the

population of P. expansum on apple surfaces.

The mode of inhibition for heat treatment is thought to involve direct inhibition of

pathogenic microorganisms, as well as induction of plant defense responses. Heat

temporarily inhibits production of volatiles by fruit (Fallik and others 1997); this is

thought to delay fruit ripening and senescence. Conway and others (2004) report that

heat treatment induces formation of fungicidal and fungistatic substances in the peel,

induces systemic plant defense response throughout the fruit, and also directly inhibits

both fungal spore germination and (radial) fungal growth.

Heat treatments are very promising for reducing storage pathogens and

controlling populations of patulin-producing molds on fruit. Heat treatment had a

beneficial effect on the long term storage quality of fruit as well. However, the effects of

heat were only curative, and heat worked best when combined with protective techniques,

such as the application of a biocontrol agent. Furthermore, the cost of heating a room to

37

38 °C may be prohibitive to small growers or producers, and although it improved

storage quality of apples, the treatment had a negative impact on the immediate eating

quality of apples.

H202

The effects of hydrogen peroxide (H2O2) on microorganisms of concern in food

processing have also been studied, using liquid phase dips or vapor treatments. H2O2 has

the benefit of decomposing completely and rapidly, leaving no residue on the produce:

plant tissues contain the enzyme catalase, which catalyzes the reaction H2O2 —> H20 + O2

(Olmez and Kretzschmar 2009).

H2O2 synthesis may be part of the systemic plant defense response that live plant

commodities mount to ward off infection. One study (Torres and others 2003) reported

that synthesis of H2O2 in apples naturally increases when fruit is challenged with fungal

pathogens, including Penicillium expansion.

In vitro studies have indicated that 5% H2O2 provided complete inhibition of P.

expansum in the agar diffusion assay (Venturini and others 2002). No studies have yet

reported significant reductions of Penicillium expansum on apples following treatment

with H2O2. Other fungal pathogens of apples have not been specifically examined. The

technology still holds promise, as H2O2 may be a viable option for reducing post-harvest

cross-contamination of fruit with fungal pathogens. Although immersing apples for 5

minutes in a solution of 27% H2O2 did not significantly reduce populations of P.

38

expansum on fruit surfaces, this solution did effect a 4 logio reduction off. expansum in

wash water (Chen and others 2004).

H2O2 has been found to inhibit fungal decay in other agricultural commodities,

aside from apple. Post-harvest treatment dips using 1.5-2.0 % H2O2 have been shown to

completely inhibit decay caused by the fungi Botrytis cinera, as well as Alternaria

alternata in eggplant and sweet pepper (Fallik and others 1994). H2O2 has been shown to

markedly decrease decay caused by Alternaria alternata and Fusarium solani in melons

(Aharoni and others 1994). However, others (Smilanick and others 1995) have found

that H2O2 dips did not reduce populations of the green mold Penicillium digitatum on

lemons, even at 15% concentration, a level of H2O2 that proved phytotoxic to the fruit.

Researchers have also investigated the inhibitory activity of H2O2 against

bacterial pathogens on apples. One study found that the effectiveness of H2O2 at

reducing populations of Escherichia coli on Golden Delicious apples was improved

slightly (to a 3-4 logio total reduction) by heating the H2O2 to 50 °C and combining it

with an acidic surfactant (Sapers and others 1999).

Post-harvest vapor treatments using H2O2 have been reported to reduce

populations of decay fungi, as well as human pathogens. H2O2 vapor reduced decay of

grapes caused by Botrytis cinerea, without affecting color or soluble solid content

(Forney and others 1991). Similar results have been obtained using vaporized H2O2 for

controlling Alternaria alternata and Fusarium solani in melons (Aharoni and others

1994), and for controlling Helminthosporium solani (silver scurf) in stored potatoes

(Afek and others 2001). H2O2 vapor has been found (Sapers and others 2003) to reduce

E. coli on apples by nearly 2 logio-

39

Aharoni and others (1994) found that H2O2 vapor treatments required a contact

time of 15-60 minutes to inhibit microbes on produce; when long treatment times or high

concentrations were used, the vapor treatment damaged melons and other fruits.

Additionally, fogging or misting setups often require expensive infrastructure and

equipment, such as the Tabor Atomizing System (Optiguide, Yokneam Illit, Israel) used

in the potato experiments described by Afek and others (2001), which is composed of

sensors, atomizers, computer interfaces, and software systems (Optiguide 2008). While

these systems are certainly appropriate, and in some cases necessary, when conducting

academic research, it is not reasonable to anticipate that such complex and expensive

systems would gain widespread use in Maine, considering the scale of agribusiness

encountered here.

Organic Acids

A number of studies have reported on the efficacy of various treatments using

short chain organic acids to reduce populations of fungi on agricultural commodities

(Singh and others 1987). The antimicrobial effects of organic acids are due to a reduction

of environmental and protoplasmic pH, and the ensuing disruption of cellular metabolism

that occurs when the undissociated acid (i.e. acid still possessing its proton) crosses the

membrane and enters the microbial cell (Jay and others 2005). Organic acids are natural

compounds, posing little or no residual hazard at the low levels required to kill fungal

spores. They are inexpensive, can be used in low concentrations, and can be used to treat

produce without handling the commodity (Tripathi and Dubey 2004).

40

Acetic acid has been studied extensively for antifungal and antibacterial activity.

In vitro antifungal activity of acetic acid (AA) against P. expansion has been described

(Venturini and others 2002), and Chen and others (2004) reported that immersing Empire

apples for 2 minutes in a solution of 2% AA completely inhibited growth of P. expansion

on fruit, and prevented the formation and accumulation of patulin within fruit.

Immersing bananas in a 0.2% AA solution inhibited fungal decay of fruit during storage

(Ethugala and Karunaratne 2002). Acetic acid is effective at inhibiting pathogenic

bacteria, as well. Karapinar and Goniil (1992) found that 0.2% AA was sufficient to

inhibit in vitro growth of the human pathogen Yersinia enterocolitica.

While glacial acetic acid seems to be effective, this is not a product that is widely

available to most apple growers or apple cider producers in the State of Maine.

Nonetheless, it may be possible to implement treatment programs utilizing lab grade

chemicals in a larger cider operation. Some large growers/processors may be well served

by a concentrated chemical that is more easily stored and transported, and which may be

used in small quantities to treat large storage areas. Another concern regarding the use of

glacial acetic acid arises from reports of phytotoxicity, even when the acid has been used

at low concentrations (Sapers and others 2003).

It is possible to provide acetic acid in a format that is readily available, making

this technology available to smaller producers, such as most cider producers in the State

of Maine. Indeed, people have been taking advantage of the antimicrobial and crop-

protective effects of acetic acid in fermented and pickled foods for thousands of years

(Pederson 1979). While most fermented foods (e.g. olives, sour pickles, sourdough

bread) contain primarily lactic acid, the primary acid in the fermented foods nata and

41

kombucha is acetic acid, produced by Gluconobacter and Acetobacter species

(Steinkraus 1996).

Acetic acid is the principle acid in vinegar, which has a long history of use in the

preservation of foods like cucumbers, beans, and fiddleheads. What we tend to describe

as "vinegary" generally refers to our perception of acetic acid. The bacteria responsible

for oxidizing alcohol into vinegar are acetic acid-producing aerobes from the

Gluconobacter, Acetobacter, and Acinetobacter genera. When these microbes are part of

the community of fermenting microbes, they lend a vinegar-like flavor to the final

product (Pederson 1979; Steinkraus 1996; Katz 2003; Jay and others 2005).

Vinegar has well documented in vitro and inplanta anti-bacterial (Karapinar and

Gonial 1992; Kanno 1998; Sengun and Karapinar 2004) and antifungal (Pasini and others

1997; Wang and others 2005b) activity, has lower phytotoxicity than glacial AA, is

readily available, is pre-diluted to a safe concentration, and is inexpensive (Sholberg and

others 2000).

Although not studied for activity against P. expansum and other toxigenic fungi,

liquid-phase vinegar treatments have been shown to be an effective method of reducing

human pathogens on the surface of apples. Wetting the surface of an apple with 5 mL of

white vinegar (5% AA), rubbing for 5 seconds, and rinsing with 200 mL of H20 reduced

populations of artificially inoculated Salmonella enterica by >5 logio (Parnell and Harris

2003). Vijayakumar and Wolf-Hall (2002) report that washing lettuce in a 35% vinegar

solution (1.9% AA) reduced Escherichia coli on lettuce by over 5 logio-

42

Others have mixed organic acids into coatings that are regularly applied to fruits,

and report a significant inhibition of Colletotrichum musae in bananas using malic, citric,

oxalic and maleic acids (Al Zaemey and others 1993).

Vapor-phase acid treatments

Organic acids can be further used in a more interesting application. Vaporized

organic acids have been used to sterilize the surface of fruit, thereby improving storage

quality and reducing losses due to rot organisms, as well as potentially reducing human

pathogens on fruit and in juice made from fruit.

It is well documented that fumigations offer a number of logistical benefits, such

as reaching all surfaces of the fruit more effectively than a liquid treatment, eliminating

the need to individually handle fruit, and reducing the possibility of cross contamination

of produce or equipment associated with dunk tanks and other washing steps (Sholberg

1998; Sholberg and others 2001).

Vaporized acids offer improved antifungal activity over liquid acids, as illustrated

by comparing the results of two sets of experiments. As described below, Sholberg and

others (2000) successfully used heat-vaporized vinegar (5.0% AA) at very low

concentrations (36.6 /uL L"1 air) to control fungal decay in stone fruit, strawberries, and

apples. However, Ethugala and Karunaratne reported that bananas immersed in full-

strength liquid vinegar (5.0% AA) displayed an increase in fungal decay causing a

decrease in shelf-life versus control fruit (Ethugala and Karunaratne 2002).

43

This disparity of results may be a result of the prevalence of the dissociated

conjugate base of AA (CH3COO) when vinegar is in the liquid phase, versus the

predominance of the more potent undissociated form of AA (CH3COOH) when vinegar

(or AA) is in the vapor phase. When in solution, organic acids tend to exist as both the

undissociated acid (RH) and a combination of free protons and dissociated conjugate

bases (H+ + R"). Contrastingly, when in the vapor phase, acids tend to exist solely as the

undissociated form (RH). In Bransted-Lowry terms, these acids have not yet donated

their proton. Undissociated acids have been shown (Eklund 1983) to have between 10

and 600 times the antimicrobial activity as dissociated acids, which is due in part to the

comparative ease with which undissociated acids cross membranes and cell walls of

bacteria and fungi.

Fumigations with numerous different acids have been reported on: treatments

with vapor of numerous short-chain organic acids (acetic, formic, and propionic) have

been found to control decay caused by Penicillium expansum on apple; P. expansion,

Monilinia fructicola, and Botrytis cinerea on cherry; M. fructicola on apricot, peach and

nectarine; and P. digitatum on orange and grapefruit (Sholberg 1998). Acetic acid has

been the most extensively researched organic acid, and seems to be quite effective.

Most research involving the post-harvest use of acetic acid vapors to reduce

fungal pathogens on fruit has been carried out by two Canadian groups: the Sholberg

group from the Pacific Agri-Food Research Station in Summerland, British Columbia,

and the Chu group from the Department of Plant Agriculture at University of Guelph,

Ontario.

44

Sholberg and Gaunce first reported on the potential in 1995, finding that

fumigations with acetic acid (AA) vapor (2.7 mg L"1) prevented post-harvest decay

caused by Penicillium expansum and Botrytis cinerea in apple and pear, with no apparent

phytotoxic effects on fruit. They also found that AA fumigations prevented B. cinerea

decay on tomatoes (Lycopersicon esculentum), grapes (Vitis vinifera), and kiwifuit

(Actinidia deliciosa), and prevented decay caused by Penicillium italicum on oranges

{Citrus sinensis). Importantly, they showed that AA fumigations not only inhibited

germination of B. cinerea spores, as reported for H2O2 fumigations, but also killed all B.

cinerea spores (Sholberg and Gaunce 1995). Thus, fumigation with AA may be able to

provide a measure of lasting protection against storage diseases caused by fungal agents.

Sholberg, Reynolds, and Gaunce (1996) continued to work with vaporized AA,

finding that a 30-minute fumigation of table grapes {Vitis vinifera) with AA vapor (2.7

mg L" ) controlled rot caused by Botrytis cinerea and Penicillium expansum as effectively

as did fumigations with SO2 (6.0 mg L"1). AA fumigation did not produce any quality

deficiencies in the treated fruit, nor did any AA residue remain on the fruit throughout

storage. Applicability and relevance of this study were increased by the researchers'

choice to fumigate grapes stored in lugs similar to those used commercially.

Moyls, Sholberg, and Gaunce (1996) went on to confirm and expand upon these

results, using other fruit. They reported that fumigating with AA vapor (8.0 mg L"1) after

inoculating fruit with Botrytis cinerea (10 CFU mL" ) protected grapes and strawberries

that were stored under a modified atmosphere for up to 74 days and 14 days respectively,

a 200-300% improvement in shelf life versus untreated fruit. The authors suggest that

45

these and previous results indicate fumigation with AA may be a suitable alternative to

sulfur dioxide fumigations for controlling decay in table grapes.

Chu, Liu, Zhao, and Tsao (1999) then reported on using AA vapor to control

Botrytis cinerea on sweet cherries {Prunus avium). Following inoculation with spores of

Botrytis cinerea, fruit was fumigated for 25 minutes with AA (30 mg L" ), and was then

placed into cold storage for 10 weeks. AA fumigated fruit exhibited 83% less incidence

of rot than untreated fruit. The authors also found that the AA fumigation had no

significant effect on fruit quality, as measured by total soluble solids, titratable acidity, or

incidence of stem browning.

In another study, Sholberg, Cliff, and Moyls (2001) examined AA fumigation of

apples under near-commercial settings, using a greater volume of apples, and storing and

fumigating fruit in standard wooden or plastic apple boxes. Again, they showed the

effectiveness of AA fumigation at reducing apple decay caused by Penicillium expansum.

They also compared the effectiveness of AA fumigation to that of a thiabendazole dip

(450 //g L"1), finding that the AA fumigation was as effective as the synthetic fungicide at

preventing post-harvest decay caused by P. expansum. Finally, this study reported that

the AA fumigation neither affected apple quality, nor imparted a "vinegar aroma".

The Chu group then confirmed the effectiveness of AA fumigations (6 mg L"1) at

significantly reducing rot caused by Penicillium expansum in sweet cherries {Prunus

avium). They reported no reduction in the incidence or severity of rot caused by

Monilinia fructicola on sweet cherry (Chu and others 2001). Later work by this group

reported reduction, but not total control, of brown rot caused by M. fructicola on apricot

46

(Prunus armeniaca) and plum (Prunus salicina), following AA fumigations (4.0-8.0 mg

L"1) (Liu and others 2002).

Sholberg has continued to report on the effectiveness of AA fumigations at

reducing rot caused by Botrytis and Penicillium on pears (Sholberg and others 2004), and

reducing common bunt (caused by Tilletia tritici and T. laevis) on wheat (Sholberg and

others 2006).

It has been reported (de Pooter and others 1984) that AA vapors induce ripening

of pre-climacteric Golden Delicious apples. No other reports confirm this observation,

but it is an interesting result, and may illuminate mode of action of acetic acid vapor.

Also, vaporized AA may control bacterial pathogens; Sapers and others (2003)

achieved a 3.5 logio reduction of E. coli on apple slices by fumigating for 30 minutes

with vaporized AA (300 mL liquid glacial AA L"1 air). Greater reductions (>5 logio)

were possible, when treatments used higher concentrations of AA and/or longer exposure

times, but these treatments damaged fruit. While the use of these higher concentrations

of AA may not be appropriate for the reduction of pathogens on fancy eating apples,

treating fruit with AA vapor may provide an added measure of protection for cider

producers who do not pasteurize, and would like to reduce the risk of potential pathogens

that has been sometimes associated with consuming raw juice.

Delaquis, Sholberg, and Stanich (1999) used AA vapor to disinfect mung bean

seeds; the authors were able to reduce populations of pathogenic bacteria {Salmonella

Typhimurium, Escherichia coli 0157:H7, and Listeria monocytogenes) to undetectable

levels (3-5 logio reductions) by fumigating mung bean seeds for 12 hours with AA (242

fi\ L" air). Although not widely adopted, this method may provide added protection for

47

producers of bean sprouts, a notorious vector for foodborne illness (McCabe-Sellers and

Beattie 2004; Greig and Ravel 2009).

A significant disadvantage to using vapor-phase acid treatments is the potential

corrosiveness of acid vapors to steel and copper (Sholberg 1998; Sholberg and others

2000).

Vapor-phase vinegar treatments

As mentioned above, while AA has documented efficacy, it is not a product that is

widely available to most cider producers or apple growers, especially the smaller

producers in the State of Maine. However, vinegar is widely available, inexpensive (it

could even be made with excess cider), has a long history of use and familiarity, is non­

toxic at appropriate concentrations, and may be used in organic processing. As

mentioned above, a significant drawback to the use of vaporized vinegar is corrosiveness.

To investigate the potential utilization of vaporized vinegar as a fungicide, Sholberg has

published a number of articles describing the use of vinegar vapor to reduce post-harvest

decay of fruits, including apples.

Sholberg and others (2000) used several common vinegars (apple cider, balsamic,

brown rice, malt, raspberry, red wine, white wine, and white), containing 4.2%-6.0% AA,

to fumigate stone fruits, strawberries, and apples that had been inoculated with Monilinia

fructicola, Botrytis cinerea and Penicillium expansum, respectively. Very small volumes

of all vinegars reduced the incidence of fungal decay in all fruit. Heat vaporization of

white vinegar (5.0% acetic acid), at a concentration of 36.6 /uL L"1 of air (approximately 1

48

quart of vinegar for a 1000 ft room), completely controlled Penicillium expansum rot in

four apple cultivars; other vinegars were found less effective on apple. The authors

reported a dose-response relationship between the incidence of lesions on apple surfaces

and the duration of vinegar fumigations, with the survival of fungi approaching zero after

about 6 hours. They also reported that increasing the concentration of conidia on the

apple surface reduced the efficacy of vinegar vapor.

Although Sholberg and others were successful, the use of vinegar vapor to control

post-harvest fungal rots on other fruits was not immediately replicated. Wang (2003)

reported that raspberries treated with vinegar vapor did not differ from control fruit

regarding incidence or severity of decay caused by Botrytis cinerea, Rhizopus stolonifer,

or Cladosporum herbarum. Using the same methodology, Wang, Chanjirakul, and others

(2005a) assessed the effects of vinegar vapor on post-harvest fungal decay of papaya, and

found "no beneficial effect" associated with the fumigation.

A difference in technique used by the groups may provide insight into the failure

of the Wang trials. Sholberg and others (2000) heated the vinegar to vaporize it, rapidly

and reliably producing a predictable concentration of volatilized acid within the

fumigation chamber. Wang sealed fruit and a small beaker of vinegar in a container,

relying on spontaneous volatilization of the acetic acid over a 14 hour fumigation period.

The Sholberg group (2000) trialed both a wick mechanism for a cold vaporization (with

moderate success), and a system for heat vaporizing the vinegar (with significant

success). Apparently, the wick mechanism did not volatilize sufficient liquid, and the

concentration of acetic acid within the chamber was not sufficient to control fungal

growth in the Wang experiments.

49

Peracetic acid

Peracetic acid (CH3COOOH) is an equilibrium mixture of H2O2 and CH3COOH

(CH3COOH + H202 <-• CH3COOOH + H20), variously referred to as Hydrogen

Peroxyacetic Acid, Peroxyacetic acid, or Peracetic Acid (PAA). For at least twenty

years, PAA has been heralded as a potential new "industry standard" sanitizer by the

brewing and dairy industries (Baldry and Fraser 1988; Lenahan 1992; Orth 1998). PAA

is a viricide, bactericide, fungicide, and sporicide, with little corrosivity; as such, it is

highly valued by the hospital and health service industry for disinfecting equipment

(Vizcaino-Alcaide and others 2003).

One particularly important advantage of using peracetic acid (PAA) to sanitize

equipment is that PAA is a strong oxidizer, and therefore may disrupt and detach biofilms

from food processing or health care equipment surfaces (Exner and others 1987; Kumar

and Anand 1998). Whether PAA can disrupt biofilms on produce is unclear, as this type

of research has yet to be carried out.

Henoun Loukili and others (2006) have reported that the ability of PAA to disrupt

biofilms may be actually due to surfactants or detergents found in the commercial

preparation, since some preparations that lack detergents may actually induce biofilm

formation in certain cases.

Numerous trials have demonstrated the in vitro antimicrobial ability of PAA

against storage rots and human pathogens that may be found on produce or equipment

(Baldry 1983; Exner and others 1987; Brinez and others 2006; Henoun Loukili and others

2006). However, there is not much literature that describes inplanta or in situ studies,

50

and the use of PAA directly on produce has been investigated for only a few foods.

There are no studies looking specifically at the use of PAA to control Penicillium species

on fruits, and limited studies have looked specifically at reducing fungi on apples, with

minimal success.

Immersion of apple slices for 5 minutes into a solution of PAA (80 ppm) has been

found to inhibit growth of yeasts and molds (species not identified) for approximately 11

days of storage, after which the PAA treatment significantly increased yeast and mold

counts on fruit surfaces (Wang and others 2007). Combining PAA with calcium

ascorbate (often used in fresh-cut apple processing to reduce browning) was even less

effective, and was found to "facilitate favorable conditions for microbial, growth".

Antifungal activity of PAA against important storage rot fungi has been reported

in other fruits (Mari and others 2004). Stone fruits (sweet cherry, apricot, peach and

nectarine) immersed for 1 minute in a solution of PAA (125 ppm) exhibited reduced

brown rot caused by the fungus Monilinia laxa. Eight minutes of immersion in a solution

of 250 ppm PAA completely controlled soft rot caused by the fungus Rhizopus stolonifer.

Another study (Martinez-Sanchez and others 2006) supports the contention that

PAA may facilitate microbial growth during storage. Washing arugula leaves in a

solution of PAA (300 ppm) promoted the growth of fungi on the produce during storage

in a low O2, high CO2 modified atmosphere.

Although the literature regarding the use of PAA to reduce fungal storage rots and

plant pathogens is paltry, there is a wealth of information documenting the efficacy of

PAA at controlling bacterial human pathogens. It is important to keep in mind that broad

spectrum antimicrobials are highly desirable for numerous reasons. They allow

51

producers to simultaneously protect consumers by reducing populations of human

pathogens, and increase the value of their crop by protecting produce against storage rots.

If evidence exists supporting its effectiveness, there may be an increase in interest for the

use of PAA to reach a 5 logio reduction of human pathogens (i.e. for a HACCP plan).

This supporting evidence could then motivate producers to use PAA, thereby also

controlling storage rots such as patulin-producing molds.

Regarding antibacterial activity of PAA on produce, 60 ppm PAA has been

reported to decrease mesophilic bacteria by 2 logio in minimally processed vegetables

and fruits (Cherry 1999). Immersion in a PAA solution (80 ppm) has been reported to

reduce populations of Listeria monocytogenes on Iceberg and Romaine lettuce at a rate

equal to 100 ppm chlorine (Beuchat and others 2004). Immersion in a PAA solution (300

ppm Tsunami®) has been found to significantly reduce and inhibit the growth of

coliform bacteria on arugula leaves, while maintaining sensory quality and antioxidant

content (Martinez-Sanchez and others 2006). Spraying almonds with a solution

containing 500 ppm PAA has been found to reduce populations of Salmonella enterica

by almost 2 logio (Pao and others 2006). Immersing apples for 1 minute in a solution of

40 ppm PAA has been reported to reduce populations of Enterobacter sakazakii by >4-

logio (Kim and others 2006). Washing sliced apples for five minutes in a PAA solution

(80 ppm) reduced populations of Escherichia coli 0157:H7 on apples by nearly 3 logio,

more bactericidal (but also more phytotoxic) than electrolyzed oxidizing water or

chlorine (Wang and others 2007).

U.S. FDA has approved the use of PAA for direct food contact at concentrations

up to 80 ppm (21 CFR § 173.315), including OMRI approval for use on organic produce.

52

PAA degrades rapidly into H2O, O2, and CH3COOH (acetic acid), leaving little residue;

PAA does not appear to form toxic decomposition products such as aldehydes,

halogenated phenols, or bromated phenols (Evans 2000; Dell'Erba and others 2007).

PAA does corrode steel increasingly as PAA concentration increases; temperature is a

factor as well, with a maximum corrosive effect for all concentrations of PAA at 20 °C

(Qu and others 2008).

Chitosan

Chitosan is a glucosamine polysaccharide, derived via deacetylation of chitin in

crab and lobster shell, and having known fungicidal activity (Allan and Hadwiger 1979;

Hirano and Nagao 1989). Chitosan has been found to be an effective inhibitor of

Penicillium expansum in apple juice (Roller and Covill 1999). It is likely that inhibition

of P. expansum would decrease patulin content of juice or cider, and chitosan may

therefore present an alternative to chemical preservatives. Chitosan has also been found

to be directly toxic to Penicillium spp. and other fungi (Liu and others 2007a), and to

induce biochemical plant defense responses that bolster resistance to fungal disease in

tomato fruit (Liu and others 2007a) and potato (Sun and others 2008). Recently, there

has been a wealth of experimentation into applications of the antimicrobial potential of

chitosan (Devlieghere and others 2004; Bautista-Banos and others 2006; Fernandez-Saiz

and others 2009). However, chitosan is not yet approved as GRAS by U.S. FDA.

53

Plant essential oils

The antimicrobial properties of various plant essential oils are increasingly being

reported in the literature (Burt 2004). It has been hypothesized that these volatiles act by

damaging cell walls and membranes of fungi, ultimately bringing about a collapse of the

fungal structures, such as hyphae and conidia (Zambonelli and others 1996). More recent

work by Bakkali and others (2008) confirms the membrane disruption theory and

describes a collapse of membrane-associated cellular processes, such as ion pumps and

ATPases. The authors surmise that additional antimicrobial effects may be due to the

pro-oxidant activity associated with certain essential oils, some of which contain

photoactive molecules like furocoumarins, in which case oxidation is induced by light.

Due to multiple modes of activity, plant essential oils are not likely to engender

resistance, and they may offer alternatives in situations where conventional treatments

have failed. As an example, topical applications of a eucalyptus essential oil formula

were used to clear a human skin infection of methicilin-resistant Staphylococcus aureus

(MRS A) (Sherry and others 2001). Nonetheless, while development of resistance seems

unlikely, caution and appropriate use of products should be stressed; there have already

been reports of bacterial tolerance to tea tree {Melaleuca alternifolia) essential oil

(Bakkali and others 2008).

When cinnamon oil (0.1%) was added to apple juice, Ryu and Holt (1993) found

that both P. expansum growth and patulin production were completely inhibited.

Thyme essential oil (thymol) has been repeatedly shown (Chu and others 1999;

Chu and others 2001; Liu and others 2002) to effectively inhibit Penicillium expansum

54

both in vitro and on apples and other fruits, although treatments were phytotoxic at

effective concentrations.

According to a recent review compiled by Batish and others (2008), vapor of

Eucalyptus tree essential oils possess antimicrobial activity against Botrytis cinerea on

apples, as well as inplanta and in vitro antimicrobial activity against Penicillium and

Aspergillus fungi; against bacteria such as Escherichia coli, Staphylococcus aureus, and

Proteus mirabilis; and even against bacterial endospores of Clostridium botulinum and

Bacillus cereus.

Isolated from buds of the clove plant (Eugenia caryophylata, syn Syzygium

aromaticum), eugenol essential oil has recently been investigated for antifungal activity

against apple pathogens, including Penicillium expansum and Botrytis cinerea.

Volatilized oil was found to be inhibitory to mycelial growth and conidial germination of

P. expansum and B. cinerea both on apples (in plantd) and in vitro (Amiri and others

2008).

Neri and others (2006) found that trans-2-hexenal (IUPAC name (E)-hex-2-enal),

the aldehyde responsible for fresh-cut grass smell and "green notes" in foods, was the

only plant volatile out of nine tested that exhibited inplanta antifungal activity against P.

expansum growing on pears.

Later, Neri and others (2007) tested the same nine volatile compounds on stone

fruit (apricot, peach, nectarine, plum), and reported that ^ra/w-2-hexenal controlled brown

rot caused by Monilinia laxa. However, at the lowest levels that would effectively

control rot, treatment with frYWs-2-hexenal was phytotoxic to all fruit but plum, and

caused off-odors and off-flavors in all fruit.

55

Others have found that treatment of pear fruit with vaporized /ra«s-2-hexanal

increases the incidence of blue mold rot caused by Penicillium expansion (Spotts and

others 2007). Sholberg and Randall (2007) supported this finding, and reported that,

while fumigation of sound apple and pear fruits with trans-2-hexena\ vapors (2-4 mg L~

') inactivated conidia of rot fungi on fruit surfaces, in some cases reducing incidence of

rot, fumigation of wounded fruit with trans-2-hQxena\ led to an increase in rot fungi on

fruit surfaces. They also reported that, in contrast to the decline in fruit quality described

by the subjective sensory analysis carried out by the Neri and Mari group (Neri and

others 2007), physical and chemical food analyses showed no harmful effects on apple or

pear firmness, pH, titratable acidity, or soluble solids.

Aside from treating fruit with vaporized volatile plant compounds, some

researchers (Serrano and others 2005; Serrano and others 2008) have tried adding

essential oils to modified atmosphere packaging (MAP), to increase the storage quality of

fruits and reduce storage rots caused by Penicillium, Botrytis and Monilinia species. The

authors reported that, following 16 days of storage at 20 °C in a MAP that included clove

essential oil (eugenol), thyme essential oil (thymol), peppermint essential oil (menthol),

or eucalyptus essential oil (eucalyptol), treated sweet cherries {Prunus avium) exhibited a

>3 logio reduction in yeast and mold populations compared to control fruit. Treatment

with all essential oils except eucalyptol improved the storage quality of sweet cherry.

Inclusion of 0.2% lemon {Citrus limon) essential oil in glucose-Czapek's-apple

medium has been found (Hasan 2000) to completely inhibit the growth of Penicillium

expansum and Aspergillus flavus. Orange (Citrus sinensis) essential oil was nearly

equally effective, providing 90% control of P. expansum and A. flavus at 0.2%; lemon

56

essential oil was reported to control 90% of P. expansum and A. flavus at the lower

concentration of 0.05%.

In vitro antifungal activity against Penicillium expansum has been reported (Neri

and others 2006) for oregano, lemon, and cinnamon bark essential oils (carvacrol, citral,

and /rara-cinnamaldehyde, respectively); yet, these oils lacked significant antifungal

activity against P. expansum when tested on pears inplanta.

Volatile oils show promise as an alternative to fumigation with synthetic

fungicides. Although some oils have obvious shortcomings, such as phytotoxicity, many

of them are remarkably effective; nonetheless, it remains to be seen whether the use of

these oils will catch on with industry.

Biofumigation/Mycofu irrigation

Some researchers are making use of another class of volatile antimicrobials in a

rather creative way. There appears to be significant potential for mycofumigation, in

which fungi are cultured in the presence of fruit, for the purpose of exposing the fruit to

the gasses produced by the metabolizing fungi. Growing Muscodor albus on sterile

grain, in the presence of fruit, is a means of exposing the fruit to numerous volatile

compounds, some of which have fungicidal activity. Originally isolated from the bark of

Honduran cinnamon trees, Muscodor albus is an ascomycota in the Xylariaceae family.

The fungus grows as a white, sterile mycelium and is not known to produce spores or

other reproductive structures; therefore, it does not survive after depleting the food source

on which it is grown, and poses no threat of spreading (EPA 2005).

57

M. albus is reported to have an inhibitory effect on numerous storage pathogens,

even in the absence of direct contact. As it grows, M. albus releases into the air

numerous volatile organic compounds (mostly esters and alcohols) which negatively

affect the growth of storage pathogens Penicillium, Colletotrichum, Geotrichum,

Monilinia, and Rhizopus. While technically a biocontrol agent, researchers have termed

this process biofumigation or mycofumigation to specify that the crop is treated with a

fumigant, albeit one that is produced by a fungus. After numerous trials demonstrating

the potential of the fungus, M. albus is now an EPA registered bio-pesticide (EPA 2005).

Mercier and Jiminez (2004) have documented complete control of Penicillium

expansum and Botrytis cinera in apples biofumigated with M. albus fungal volatiles,

reporting that the fumigation treatments "gave excellent control of blue mold and gray

mold of apples, as well as brown rot of peaches regardless of the fumigation time or dose

tested". Mercier and Smilanick (2005) reported that one to three day M. albus

biofumigations of lemon (Citrus limon) completely controlled Penicillium digitatum and

Geotrichum citri-aurantii (the causative agents of green mold and sour rot, respectively).

More recently, Schotsmans and others (2008) reported on how the control of

fungal apple rots provided by a three day biofumigation using M. albus is affected by

modified atmosphere and temperature. Although very effective under all conditions

tested at controlling populations of fungi in vitro, when trials were conducted with live

Honeycrisp™ apples, M. albus provided variable control of fungal rots caused by

Penicillium expansum, Sclerotina sclerotiorum, and Botrytis cinerea. In planta studies

revealed that biofumigation with M. albus controlled S. sclerotiorum and B. cinerea

58

effectively at all temperatures when stored in regular air but not when stored in either of

two modified atmospheres (reduced O2 or increased CO2).

Others have found that peaches shipped with a small pad containing metabolizing

Muscodor albus significantly reduced incidence of brown rot caused by Monilinia

fructicola (Schnabel and Mercier 2006).

Biological control

Definition

Biological control is a term used to describe "the use of parasitoid, predator,

pathogen, antagonist, or competitor populations to suppress a pest population, making it

less abundant and thus less damaging than it would otherwise be" (Van Driesche and

Bellows 1996). Biological control (biocontrol) may be thought of as a third fundamental

means of controlling pests. In the same way that we may employ chemical pest control

(e.g. applying a chemical insecticide) or physical pest control (e.g. trapping insects), we

may also employ biological control. Antagonists inhibit pests by producing antibiotics or

toxins, competing for resources, parasitizing pathogenic organisms, or inducing plant

defense responses in the host (Spadaro and Gullino 2004).

Biological control may be used by some to refer to a natural predator-prey

phenomenon; for clarity, this idea is more specifically referred to as "natural biological

control". Generally, biological control is meant to be understood as the active

management of populations; it is any purposeful manipulation of the ecology of pests and

59

their natural enemies (antagonists) to control the amount or severity of damage inflicted

by pests (DeBach and Schlinger 1964; Van Driesche and Bellows 1996).

There are three broad mechanisms by which biological control is generally

deployed. The importation of exotic antagonists is possible; however, this method is not

always successful and carries the risk of incidental introduction of a new pest species, as

well as other unforeseen ecological consequences. Second, we may augment naturally

present populations of antagonists by directly manipulating the endemic population of

antagonists; for instance, we may rear and then release native antagonists en masse.

Finally, we may conserve or attract native antagonists via manipulation of the

environment; for example, we may attract pollinators by planting attractive flowers along

with the primary crop (DeBach and Schlinger 1964).

History & emergence of modern concepts

DeBach and Schlinger (1964) describe the mid-1800s introduction of the mynah

bird to Mauritius, for controlling the red locust, as the first successful instance of

biological control achieved by transferring a natural antagonist from one country to

another. Others (Legner 2008) have gone back even further, describing the use of

domestic cats to control rodent populations in ancient Egypt as an instance of biological

control.

Whether or not these historical examples are considered biological control, the

term "biological control" was likely used first in 1919, by Harry S. Smith of the

University of California, Riverside (Smith 1919), to describe what had previously been

60

called "parasitic control", "use of insect enemies", and "the biological method" (Baker

and Cook 1974). In 1947, under Smith's direction as chair, UC-Riverside's Division of

Beneficial Insect Investigations became the Division of Biological Control (Legner

2008).

However, biological control was not an idea familiar to most, until the late 1960s,

after the concept was popularized by a series of symposia held at the University of

California, Berkeley, beginning in 1963. In 1964, DeBach and Schlinger published

Biological Control of Insect Pests and Weeds, a collection of papers that detailed

investigations into the efficacy and applications of biological control. In 1965, Baker and

Snyder published the proceedings from the initial UC-Berkeley symposium on biological

control.

At this point, most of the interest in biological control surrounded soil

microbiology. Most individuals writing about biological control were soil

microbiologists, plant pathologists, and crop scientists. These scientists were interested

in modifying soil microbial ecology to prevent the persistence or incidence of soil-borne

microbes that are the causative agents of significant agricultural crop diseases.

After biological control began attracting more interest, Ordish (1967) published

what was likely the first text devoted to explaining and detailing the concepts of

biological control. Van de Bosch and Messenger (1973) then published a short, but

widely read, text on biological control, furthering awareness and sparking curiosity

among scientists.

In 1974, Baker and Cook published what has become a definitive text, Biological

Control of Plant Pathogens. The book is comprehensive, describing the roles in plant

61

pathogenesis played by host, pathogen, antagonist, and environment. Baker and Cook

thoroughly document both successful and unsuccessful attempts at using biological

agents to control plant disease. Interestingly, all the work at this point still revolves

around soil microbiology. There is a focus on manipulation of soil ecology, and

biological control is always implied to be pre-harvest, a technique of disease management

during the growing season. In their book, Baker and Cook (1974) present a thorough and

unified concept of biological control at this time:

"Biological control is the reduction of inoculum density or disease-producing activities of a pathogen or parasite in its active or dormant state, by one or more organisms, accomplished naturally or through manipulation of the environment, host, or antagonist, or by mass introduction of one or more antagonists..."

This stress on first studying and understanding, and then manipulating the ecology of

plant disease may be Baker and Cook's greatest contribution to the field of biological

control. They specified and clarified the goals of biological control, the limitations to its

application, and the broad mechanisms by which biological control works:

"Biological control rarely eliminates a pathogen... but rather reduces its numbers or its ability to produce disease; such control may be achieved with little or no reduction in population of the pathogen, or perhaps without preventing infection; for this reason, biological control tends toward stability at a low level of disease."

As mentioned above, there are numerous modes of employing biological control

within agricultural systems. Manipulations of the environment, host, or antagonist all

have a place in pest management, and may play a significant role in controlling patulin-

producing molds. However, there are many variables involved when employing

biocontrol techniques, and specialized training is often required, which currently makes

these tactics unrealistic. We will focus on the more widely familiar, popular, and well-

studied biocontrol technique of mass introductions, either of endemic or exotic

antagonists.

62

Process of developing biological control agents

Screening for potential antagonists is the first step in developing or describing

potential biocontrol agents (Droby and others 2009). Wilson and others (1993) describe

an uncomplicated method of isolating potential antagonists: after wounding and

incubating fruit for 10 days, the authors scraped wounds that showed no decay. From

these samples, they isolated frequently occurring colonies, which were further screened

for in vitro antagonistic activity against Botrytis and Penicillium species. Six isolates

were found to be effective antagonists against both Botrytis and Penicillium rots in apple.

However, as later noted by some of the authors of this paper (Droby and others 2009),

this technique generally favors quickly growing microbes, yielding control agents that

exhibit protective, but rarely curative properties against storage pathogens.

Others have tried more methodical laboratory-based screens, assessing in vitro

antibiosis, resource utilization, and other relevant factors. Searching for latent

antagonists to the Agrobacter species responsible for bacterial crown gall disease in

grapes, Bell and Dickie (1995) isolated and screened 851 epiphytic bacteria. They found

24 strains that inhibited the Agrobacteria in vitro; of these, one showed significant

potential when tested in situ.

Burr and others (1996) had similar results when they screened 931 bacteria and

yeast for inplanta antagonistic activity against Venturia inaequalis, the fungal causative

agent of apple scab, a disease that affects both fruit and trees. Of these 931 isolates,

thirty-two completely inhibited fungal germination in vitro; one-hundred six significantly

reduced the amount of apple scab that formed on seedlings {inplanta). Interestingly,

63

there was no correlation between in vitro antibiosis and ability to suppress fungal disease

in planta; only one isolate (Pseudomonas syringae) was found to both prevent V.

inaequalis conidial germination in vitro and control disease in planta at a level

comparable to the fungicide captan.

Dickie and Bell (1995) published an analysis of some nine factors (e.g. strain of

pathogen and antagonist, culture media, growth conditions) found to significantly affect

the outcome of the in vitro screening tests. This publication and others assessing

commercialization (Mathre and others 1999; McSpadden Gardener and Fravel 2002;

Montesinos 2003), safety concerns (Cook and others 1996), and the state of the art

(Shoda 2000; Janisiewicz and Korsten 2002; Krishna and McSpadden Gardener 2006;

Droby and others 2009) of biological control of plant pathogens, have established a

protocol for isolating, identifying, and then making use of endemic microbes as

biological control agents, also known as bio-pesticides or microbial pesticides. This

protocol has facilitated more exploration of and experimentation with many potential

microbial antagonists, as well as experimentation with combining different antagonists

and combining biocontrol with chemical and/or physical methods of control.

An analysis of the use of transformed, recombinant, or genetically modified

microbes is conspicuously absent in the following discussion. While these types of

microbes may play a role in the reduction of post-harvest pathogens in the future, their

mode of inhibition often involves expressing genes that code for explicitly antimicrobial

compounds (Whitten and others 1999; Janisiewicz and others 2008a), rather than

inhibiting pathogens through physical attachment, resource competition, or parasitism.

Transformed microbes often simply represent a novel delivery vector for synthetic drugs;

64

these transformed biological control agents seem more likely than natural antagonists to

engender resistance through long term use.

Pre-harvest biological control

The use of introduced microbial biocontrol agents (both fungi and bacteria have

been investigated) on fruit either pre- or post-harvest is a promising means of protecting

fruit from infestations of fungal storage rots, as well as human pathogens.

Late season field applications (e.g. using motor-driven backpack sprayers) of

bacterial {Bacillus subtilis) and fungal {Rhodotorula glutinis, Aureobasidiumpullulans,

Cryptococcus infirmo-miniatus, and Cryptococcus laurentii) antagonists reduced

incidence of storage rot caused by Penicillium expansum in apples (Leibinger and others

1997) and pears (Benbow and Sugar 1999).

Honeybees {Apis mellifera) and various kinds of bumblebees {Bombus spp.) have

been used to deliver targeted inocula of microbial biocontrol agents to flowers of fruit

plants. Experimenters set up dusting doors at the entrance of hives, and these dusters

deliver a measured unit of beneficial microbes, lyophilized or otherwise prepared, to each

bee as it enters and/or exits the hive. Utilizing bees, it has been possible to control

Botrytis cinerea in strawberries (Kovach and others 2000) and raspberries (Yu and Sutton

1997), and to control Monilinia vaccinii-corymbosi in blueberries (Dedej and others

2004).

However, applying biocontrol agents in the field is generally labor intensive, and

accurately targeting all plants is not always possible, even if the antagonists are

65

disseminated by pollinators. The use of pollinators to disseminate biological control

agents is only appropriate when application to the flower will provide effective control of

plant pathogens. Many apple growers already utilize pesticides in the field. The majority

of apple growers in the State of Maine are not organic, and do make use of at least

carefully timed and appropriately measured applications of fungicides and other

pesticides throughout the season to control scurf, scab, and other diseases that may

reduce yield or degrade apple quality.

Pre-harvest applications of microbial biocontrol agents may be limited by weather

events or trends (Shtienberg and Elad 1997). Even without a storm or high winds, harsh

environmental conditions may present a hurdle to employing biological control

successfully in the field. Pre-harvest microbial biocontrol agents must be tolerant of high

intensity sunlight, temperature fluctuations, variable humidity, and low availability of

nutrients (Ippolito and Nigro 2000).

Post-harvest biological control

Comparatively, post-harvest biocontrol is directed immediately at the produce, is

generally uninfluenced by weather, and has been shown to be an effective alternative to

post-harvest applications of synthetic fungicides. Post-harvest biocontrol can be carried

out under exact environmental conditions rather than being subject to weather conditions,

allows for highly directed and effectively targeted control of pathogens, and is a more

cost effective method of utilizing often expensive biological control products and

procedures (Wilson and Pusey 1985; Pusey 1996; Sholberg and Conway 2001).

66

Post-harvest biological control agents inhibit pathogens through the same means

as all biological control agents; they compete for resources, produce antibiotics,

parasitize the pathogen (often by producing lytic enzymes or attaching to hyphae), or

induce plant defense responses and resistance mechanisms in the host (Spadaro and

Gullino 2004). Disease suppression is usually accomplished through more than one

mechanism of action (Droby and others 2000; Droby and others 2009). Post-harvest

biological control has been shown to protect against further infestations of pathogens,

prevent growth of endemic pathogens, and even directly reduce populations of endemic

pathogens in some cases.

The pathogen population existing on fruit surfaces at the time of treatment

dramatically influences the rate of success of biological control. Antagonists may not be

able to control a pathogen whose numbers are too large (Sholberg and Conway 2001).

Antagonists usually cannot eradicate latent infestations, nor can they completely sanitize

fruit surfaces; once a population of pathogens is established on produce, biocontrol

agents are generally of little help (Janisiewicz and Korsten 2002; Krishna and

McSpadden Gardener 2006).

Wilson and Pusey may have been the first to publish results from post-harvest

applications of biological control agents. The authors describe complete control of

Monilinia fructicola when Bacillus subtilis was mixed into the wax normally used to coat

peaches (Wilson and Pusey 1985). Others followed suit, and soon many researchers were

scouring the surfaces of fruit and leaves to find antagonistic microbes which could be

isolated and utilized as post-harvest biocontrol agents (Janisiewicz 1987, 1988, 1989;

67

Wisniewski and Wilson 1992; Jijakli and others 1993; Bell and others 1995; Burr and

others 1996).

Fungal post-harvest biological control agents

A number of fungal post-harvest biological control agents have been explored for

their potential to protect apples against fungal storage rots, including Penicillium

expansum. For the most part, these fungi are yeasts, or yeast-like anamorphs of

filamentous ascomycota or basidiomycota, belonging to numerous genera.

Rhodotorula glutinis

Immersing apple or pear in a solution containing the basidiomycetous yeast

Rhodotorula glutinis (10 cells mL" ) has been reported to control Penicillium expansum

(Chand-Goyal and Spotts 1997; Calvente and others 1999; Castoria and others 2005).

More recent work (Zhang and others 2009) confirms that R. glutinis can control P.

expansum in apple, and also found that control with R. glutinis does not adversely affect

quality parameters of treated apples. The closely related Rhodotorula aurantiaca

(previously classified as R. glutinis var. aurantiaca) has also been found to control

Penicillium expansum in apples (Chand-Goyal and Spotts 1996).

Evidence suggests that R. glutinis controls pathogen growth by competing for

nutrients. At low concentrations of iron, R. glutinis produces rhodotorulic acid, a

siderophore (low molecular weight ferric chelating agent), which competes with fungal

68

pathogens for iron at the wound site, thereby inhibiting pathogen hyphal growth and

spore germination (Chand-Goyal and Spotts 1997; Calvente and others 1999; Zhang and

others 2007). If iron is in high concentration, the biocontrol activity of R. glutinis may be

reduced; concomitantly, the addition of excess rhodotorulic acid may increase the

effectiveness of the antagonist. The addition of supplemental rhodotorulic acid reduced

the inoculum density of R. glutinis required to control pathogens (Calvente and others

1999). Compared to the excretion of antifungal compounds, these modes of action are

desirable in a biological control agent meant as an alternative to synthetic fungicides, as

they are not likely to engender resistance through continued usage (Chand-Goyal and

Spotts 1996; Chand-Goyal and Spotts 1997).

R. glutinis may have additional appeal to those looking for a patulin control

organism. In addition to inhibiting fungal growth, R. glutinis appears to partially digest

patulin. Castoria and others (2005) found that apples treated with R. glutinis showed a

significant decrease in recoverable patulin. Upon further analysis of treated apples, the

researchers observed the appearance of two major spots on thin-layer chromatography

plates, suggesting that R. glutinis somehow inhibits production or accumulation of

patulin, potentially by metabolizing it.

Pichia spp.

The ascomycetous yeast Pichia anomala controls P. expansum and Botrytis

cinerea in apple, although details concerning treatment method were unavailable (Jijakli

and others 1993). While mode of inhibition for P. anomala has not been studied, that of

69

a closely related species, P. membranefaciens, has been described (Chan and Tian 2005).

P. membranefaciens was investigated, in planta and in vitro, for potential to control P.

expansum and other apple pathogens. Chan and Tian did not make an attempt at

duplicating commercial conditions; rather, the researchers were concerned with exploring

the potential and describing the mechanisms of action for P. membranefaciens. They

found that P. membranefaciens secretes hydrolytic enzymes (chitinases and glucanases)

and strongly attaches to hyphae of Penicillium expansum, Rhozopus stolonifer, and

Monilinia fructicola. The authors reported evidence that the attachment process was at

the root of the mode of antagonism, and that this process was coordinated by cell-surface

protein-signal recognition between antagonist and pathogen.

Cryptococcus spp.

The basidiomycetous yeast Cryptococcus albidus, originally isolated from peach

fruit, was found (Fan and Tian 2001) to completely control both Penicillium expansum

and Botrytis cinerea in apple, when applied at a concentration of 10 cells mL"1. Control

was improved with the addition of CaCi2, facilitating control with concentrations of

antagonist as low as 10 CFU mL" . However, control was reduced if the antagonist was

not applied prior to the pathogen, indicating that this species has predominantly

protective capacity, and little curative ability.

Investigating mechanisms of activity, Fan and Tian (2001) found that a yeast

culture filtrate of C. albidus was unable to provide control of the pathogens. This result

indicates that competition for resources and/or direct attachment to the pathogen are the

70

likely modes of action, rather than secretion of inhibitory compounds. Recently, Chan

and Tian (2005) supported this mechanism of control and elaborated; antagonism

involves the secretion of hydrolytic enzymes (endo-chitinases) and the formation of an

extracellular matrix. This matrix then surrounds and facilitates attachment to the terminal

end of pathogenic hyphae, physically blocking pathogen growth. Chan and Tian also

surmised that the interaction was coordinated by protein-signal recognition between

antagonist and pathogen.

The closely related basidiomycetous yeasts Cryptococcus infirmominiatus (also

infirmo-miniatus) (Chand-Goyal and Spotts 1996; Chand-Goyal and Spotts 1997) and C.

laurentii (Chand-Goyal and Spotts 1996; Chand-Goyal and Spotts 1997; Qin and Tian

2005; Conway and others 2007) have individually been found to inhibit Penicillium

expansum in apple and cherry, when applied post-harvest at a rate of 107 CFU mL"1.

Authors reported the yeasts were not inhibited by a modified atmosphere (1% O2 99%

N2) (Chand-Goyal and Spotts 1997; Conway and others 2007).

For some apple growers or cider producers deciding on a pest-reduction strategy,

the only concern is effectiveness. In recent history synthetic fungicides have often met

this concern adequately, but more recently their effectiveness has been decreasing. Post-

harvest biological control provides an alternative to synthetic fungicides, but it also may

effectively supplement these fungicides, improving their effectiveness and possibly

preventing the development of resistance.

Chand-Goyal and Spotts (1997) reported improved control when combining C.

infirmo-miniatus and a half-strength dose of the synthetic fungicide thiobendazole (264

Hg mL"1) (91% control) or using both C. infirmo-miniatus and C. laurentii (84% control).

71

Both of these biocontrol treatments yielded significantly better control than was obtained

with thiabendazole treatment at the commercially recommended high rate of 528 /xg mL"1

(79% control).

The closely related Cryptococcus laurentii has been investigated for control of P.

ft S

expansum, as well. Post-harvest applications of a suspension of C. laurentii (10 - 10

CFU mL" ) controlled P. expansum in apples (Chand-Goyal and Spotts 1996; 1997; Vero

and others 2002; Yu, Li and others 2007) and other fruits (Benbow and Sugar 1999; Qin

and Tian 2005). Control of P. expansum on apples was improved when C. laurentii was

combined with the plant hormones salicylic acid (Yu and others 2007a) or gibberelic acid

(Yu and Zheng 2007). Control was also improved when C. laurentii was combined with

the glucosamine polysaccharide chitosan (Yu and others 2007b). Post harvest control of

P. expansum on sweet cherry was improved by including silicon in the treatment (Qin

and Tian 2005). The authors report that silicon has strong effects on its own, stimulating

antagonists and inhibiting pathogens, but also may play a role in inducing plant defense

responses, and therefore acts synergistically when applied with a biological control agent.

Silicon inhibits growth of a number of rot fungi and stimulates the growth of C. laurentii

on fruit during long-term storage (>48 days). Additionally, silicon induces the

production of phenylalanine ammonia lyase (PAL), polyphenol oxidase (PPO), and

peroxidase (POD). These enzymes are involved in the biosynthesis of important plant

defense molecules (phenolics, phytoalexins, and lignins), which are highly toxic to

invading microbes. These defense responses are responsible for browning reactions at

wound sites; therefore, treatment with silicon may be contraindicated in certain

72

circumstances where avoidance of excessive browning is paramount, such as for fancy

eating apples.

C. laurentii also appears to consume butyl acetate, acetate esters, and other

volatiles emitted by apple fruits during storage, which have been found to cue conidial

germination of Botrytis cinerea (Filonow 2001) and Penicillium expansum, according to

more recent unpublished data from Filonow. Volatiles released by fruit in storage appear

to stimulate only pathogenic fungi to conidial germination, and when antagonists

consume some of these volatiles as a foodstuff, their ability to outcompete or otherwise

inhibit pathogens is augmented.

Metschnikowia pulcherrima

The ascomycetous yeast Metschnikowia pulcherrima has been found to

effectively control Penicillium expansum on Golden Delicious apples and in vitro

(Leverentz and others 2000). Janisiewicz and others (2001) found that, without

7 1

producing a fungicide, M. pulcherrima (10 CFU mL" ) inhibited P. expansum spores

present in a mixed suspension. The antagonist reduced mold growth in the solution and

colonized fruit surfaces, significantly reducing P. expansum rot in apples during three

months of cold storage. M. pulcherrima is reported to have variable tolerance to the anti-

scald chemical DPA; of the eight strains tested, all showed sensitivity to DPA, though

inhibition of the antagonist was not generally sufficient to preclude simultaneous use.

These findings are relevant since DPA has been the most important post-harvest

73

treatment of apples for growers who do not store fruit under controlled or modified

atmosphere, and for growers who store fruit longer than three months.

Neither M. pulcherrima culture filtrate nor autoclaved M. pulcherrima cells

provided control of fungal pathogens (Spadaro and others 2002), indicating that the mode

of inhibition is likely resource competition. This idea is supported by recent work

(Saravanakumar and others 2008) that suggests competition for iron at the wound site in

planta; addition of iron to nutrient media reduces in vitro ability of M. pulcherrima to

control P. expansum.

Interestingly, M. pulcherrima may also be able to control the growth of human

pathogens on fruit; the fungus appears to inhibit L. monocytogenes and Salmonella

enterica on apples (Leverentz and others 2006).

As for most post-harvest biological control agents, M. pulcherrima does not

sufficiently control latent populations of plant pathogens. Control may be improved,

however, by combining biocontrol with another control step.

As previously mentioned, Leverentz and others (2000) found that holding apples

at 38 °C (85% RH) for four days was effective at reducing or eliminating incidence of P.

expansum rot during storage. However, heat provided no residual treatment, and if the

fruit was challenged at any time, they were susceptible to infection by fungal pathogens.

To compete with synthetic fungicides, a technique must be able to both cure and protect

against fungal infestations. The authors had greatest success reducing incidence of

storage rot when they combined heat treatment, an eradication step, with post-harvest

microbiological control (M pulcherrima or Pseudomonas syringae), a protection step.

This work was later duplicated by Leverentz and others (2003), again with success.

74

Spadaro, Garibaldi, and others (2004) found that pre-treatment with a similar regime of

o i

heat improved the control capability of M. pulcherrima (10 CFU mL" ), including

control of latent molds. Conway and others (2004) also found that a multi-day treatment

using moderate heat significantly improved M. pulcherrima control of established

populations of P. expansum on fruit. These findings were later repeated, and heat

treatment was found to improve control by M. pulcherrima and by C. laurentii (Conway

and others 2005).

Although effective for the control of fungal pathogens on storage apples, heat

treatments may require large input in the form of capital or equipment, or be prohibitively

expensive to operate for some producers. Comparatively, sodium bicarbonate (SBC,

NaHCOs) is inexpensive, readily available, and may provide an alternative method of

fruit surface sanitation when included as part of a combinatorial approach to post-harvest

disease reduction.

Conway and others (2004) found that immersion of fruit in a suspension of 1%

SBC significantly improved the control of established populations of P. expansum by M. 7 1

pulcherrima (10 CFU mL" ). Spadaro, Garibaldi, and others (2004) confirmed that

immersion of apples into a solution of SBC (5%) prior to treatment with M. pulcherrima Q 1

(10 CFU mL" ) improved control of P. expansum. Janisiewicz and others (2005) also

found that immersion in a solution of 2% SBC improved the ability of M. pulcherrima 7 1

(10 CFU mL" ) to control P. expansum on apples.

Conway and others (2005) then reported that combining biocontrol agents can

improve efficacy of P. expansum control. They described how a combination of M.

pulcherrima and C. laurentii controlled P. expansum rot during four months of cold

75

storage significantly better than did M. pulcherrima alone, and notably (though not

significantly) better than C. laurentii alone. The combination of immersion in a solution

of 2.0% SBC and treatment with a post-harvest microbiological control agent eliminated

decay in most cases; while not a statistically significant improvement, complete control

was not possible in any other treatment that did not involve a heat treatment.

Later work synthesized some of the above findings and reported that under

laboratory conditions, P. expansum rot was completely controlled by a combination of

immersion in 2.0% SBC, treatment with a combination of M. pulcherrima and C.

laurentii, and storage in a controlled atmosphere (1.4 kPa O2, 3 kPa CO2) (Conway and

others 2007). The authors later confirmed these results under simulated commercial

settings (Janisiewicz and others 2008b).

Candida oleophila

The ascomycetous yeast Candida oleophila, is an EPA registered pesticide that

controls post-harvest storage diseases of numerous fruits (EPA 2000a). C. oleophila (106

CFU mL" ) inhibits Penicillium expansum both in apples and in vitro; biocontrol activity

was augmented by adding CaCL; to the formulation of the biocontrol agent (Wisniewski

and others 1995). Recent work confirms C. oleophila control of P. expansum on apples

o t

in storage: immersing fruit in a solution of C. oleophila (10 CFU mL" ) was found to

reduce incidence of P. expansum rot in apples during 3 weeks of cold storage (Droby and

others 2003).

76

Work by others (El-Neshawy and Wilson 1997) has shown that combining the

o t

peptidic bacteriocin nisin (0.4%) with C. oleophila (10° CFU mL"') improves control of

P. expansum. The authors report that wound ecology and populations dynamics are

manipulated by the inclusion of nisin, and the bacteriocin creates an environment

favorable to C. oleophila but inhospitable to P. expansum.

As with other post-harvest biocontrol agents, C. oleophila's ability to cure and

protect fruit against P. expansum was improved by immersing the fruit in a solution of

sodium bicarbonate before applying the antagonist (Droby and others 2003).

The mode of activity of C. oleophila is thought to involve induction of plant

defense responses (de Capdeville and others 2002) and production of extracellular lytic

enzymes, such as chitinases, proteases, and glucanases (Bar-Shimon and others 2004).

Interestingly, C. oleophila may also inhibit L. monocytogenes and Salmonella

enterica on apples (Leverentz and others 2006).

Candida saitoana

Post-harvest treatment of fruit with a suspension of the ascomycetous yeast

o i

Candida saitoana (10 CFU mL" ) controls Penicillium expansum in apples comparably

to thiabendazole (El-Ghaouth and others 2001). This group and others found that control

could be significantly improved by delivering the antagonist with either glycochitosan

(El-Ghaouth and others 2000a; El-Ghaouth and others 2000b) or the glucose analog 2-

deoxy-D-glucose (El-Ghaouth and others 2001), although evidence suggests that 2-deoxy-

77

D-glucose (DOG) may increase patulin production in those fungi that do survive (Kazi

and others 1997; Paterson 2007).

Candida saitoana appears to control Penicillium expansum and other rot fungi

through multiple modes of action. C. saitoana severely injures the hyphae of rot

organisms (El-Ghaouth and others 1998) and induces systemically expressed plant

defense responses (de Capdeville and others 2002), such as chitinase and glucanase

production (El-Ghaouth and others 2003). Therefore, C. saitoana has the potential for

preventing both the growth of pathogens and the development of necrotrophic rot that is a

result of the cytological damage associated with a high load of fungal rot organisms.

Candida sake

Post-harvest treatment of apples with a suspension of the ascomycetous yeast

Candida sake (10 CFU mL" ) controls P. expansum (Jijakli and others 1993; Vinas and

others 1998; Teixido and others 1999; Usall and others 2001) and Botrytis cinerea (Jijakli

and others 1993) in stored fruit.

The effectiveness of C. sake as a biocontrol agent has been confirmed under

various controlled atmospheres that simulate commercial conditions (Usall and others

2000). C. sake is also compatible with the important post-harvest scald inhibitor DPA

(Usall and others 2001).

C. sake reduces incidence and severity of Penicillium expansum rot more

effectively at 5 °C than 25 °C (Vero and others 2002). Recent work (Morales and others

2008c) suggests that C. sake may effectively control P. expansum in cold storage, but

78

once apples are removed from refrigeration, P. expansion mycelial growth may exceed

that of untreated apples. Patulin levels may then correlate to a similar curve, being

inhibited during cold storage, but quickly rising to levels in excess of untreated fruit after

a short time at ambient temperatures.

The method of pathogen inhibition may involve competition for space and

nutrients at the wound site (Vinas and others 1998). C. sake produces neither antibiotics

nor chitinases (Usall 1995; Vero and others 2002), and it appears that nitrogen limits the

growth of both pathogen and antagonist (Vero and others 2002), suggesting competition

for nitrogen may play a role in wound ecology.

A ureobasidium pullulans

The post-harvest treatment of apples with a suspension of the yeast-like

ascomycota Aureobasidium pullulans (10 CFU mL" ) controls P. expansum on fruit

(Ippolito and others 2000; Bencheqroun and others 2007).

The mode of activity of A. pullulans is multifaceted, involving competition for

amino acids and sugars at the wound site (Bencheqroun and others 2007), stimulation of

plant defense responses that include production of glucanase, chitinase, and peroxidase

(Ippolito and others 2000), and production of aureobasidin A (AbA), which is an

antifungal cyclic depsipeptide antibiotic that has been found to be broadly inhibitory to

fungi and protozoa (Liu and others 2007b).

Interestingly, A. pullulans is the name given to the asexual form of, or anamorph

of, Discosphaerina fulvida a fungal pathogen of flax and cotton (Sivanesan 1990). The

79

closely related D, fagi may inhibit L. monocytogenes and Salmonella enterica on apples

(Leverentz and others 2006).

Bacterial post-harvest biological control agents

Compared to the fungal species investigated, far fewer bacterial species have been

explored for their potential to biologically control P. expansum and other patulin-

producing molds on apples in storage.

Pantoea spp.

Pantoea, a genus within the family Enter obacteraceae, includes a number of

species of gram negative rods, many of which were formerly included in the genus

Erwinia (Mergaert and others 1993). While Erwinia includes numerous plant pathogens

(Agrios 2005), and certain Pantoea species may act as plant pathogens (see below), a

number of Pantoea species have been investigated for antagonism against plant

pathogens.

Minimally effective at controlling P. expansum on apples, Pantoea agglomerans

has nonetheless been reported to effectively control patulin accumulation in apples when

applied post-harvest at the relatively low concentration of 104 CFU mL'1 (Morales and

others 2008b). Others (Nunes and others 2001) have found P. agglomerans successful at

controlling post-harvest P. expansum rot in pear, as well as numerous fungal storage

80

pathogens of stone fruit (Bonaterra and others 2003), citrus (Canamas and others 2008),

and grape (Trotel-Aziz and others 2008).

Pantoea ananatis (syn. P. ananas) (10 CFU mL" ) has been found to control P.

expansum in apples stored up to two months at refrigerated temperatures (Torres and

others 2005). Although P. ananatis was effective in air, control of P. expansum was

improved when fruit was stored under an atmosphere with low O2 concentration. Recent

work (Usall and others 2008) described an integrated treatment combining the application

of P. anantis and the immersion of fruit into 3% SBC to cure and protect against

Penicillium digitatum in orange and lemon fruit.

Certain strains of P. ananatis are at least opportunistically phytopathogenic, for

instance, taking advantage of insect damage to parasitize a crop (Gitaitis and others

2003). P. ananatis has been found to be the causative agent of center rot in onions

(Gitaitis and Gay 1997; Walcott and others 2002; Gitaitis and others 2003), leaf blotch

disease in Sudanese sorghum (Azad and others 2000), and brown stalk rot (Goszczynska

and others 2007) and leaf spot (Paccola-Meirelles and others 2001) disease in maize.

Additionally, there has been at least one report (Baere and others 2004) of human

pathogenicity associated with strains of P. ananatis, which may opportunistically infect

individuals with a compromised immune system.

Rahnella aquatilis

Another member of the Enterobacteraceae, the epiphytic gram-negative rod

Rahnella aquatilis may be isolated from fruit and leaves of apples, and is often found in

81

water. R. aquatilis antagonizes Penicillium expansum both in vitro and on apples (Calvo

and others 2007). Complete control was obtained when fruit treated with a solution ofR.

aquatilis (106 CFU ml/1) was stored at 15 °C. When treated fruit was stored at 4 °C,

control of P. expansum was significant, though not complete.

This bacterium may possibly pose a health risk. While the above authors reported

that the strain of R. aquatilis used in their experiments exhibited no acute animal toxicity,

some strains of R. aquatilis are opportunistic human pathogens, and there have been

reports of R. aquatilis bacteremia in hospital patients (Menasalvas and others 1996;

Caroff and others 1998; Boukadida and others 1999). There may be an elevated risk for

individuals with compromised immunity: HIV/AIDS patients have been reported to be

susceptible to opportunistic infections of R. aquatilis (Funke and Rosner 1995).

Pseudomonas spp.

The family Pseudomonadaceae is comprised of five genera, including the truly

ubiquitous and highly variable Pseudomonas genus (Moore and others 2006).

Pseudomonas species have been used for bioremediation, synthesis of various products,

and plant growth stimulation, in addition to biological control of plant pathogens (Galli

and others 1992). Pseudomonads, including strains of P. syhngae, may act as plant

pathogens in some cases. However, the strains that have been studied and written about

have not been found to pose a significant threat to fruit.

Following a screen of more than 800 potential antagonists isolated from apple

tissue, Janisiewicz (1987) isolated an unidentified Pseudomonas species as a potential

82

biological control agent of P. expansum on apples and in vitro. Janisiewicz went on to

successfully inhibit P. expansum on apples by treating fruit with a suspension of P.

syringae (108 CFU mL"1) (Janisiewicz 1988). The bacterial antagonist was effective, but

only if applied at least 72 hours before fruit was challenged with the fungal pathogen.

Janisiewicz found that if the bacterial antagonist was applied with a second antagonist, a

yeast shown to inhibit Botrytis cinerea in apples, the ability of the Pseudomonas species

to control Penicillium expansum was improved.

More studies confirmed the potential of P. syringae to control P. expansum in

apples (Janisiewicz and Peterson 2004; Errampalli and Brubacher 2006), to control P.

expansum and B. cinerea in apples (Janisiewicz and Jeffers 1997; Janisiewicz 1998; Zhou

and others 2001; Mikani and others 2008), and to control P. expansum and other post-

harvest fungal diseases in pears (Janisiewicz 1989; Janisiewicz 1992). Of particular

interest to most apple growers and cider producers, Janisiewicz and Jeffers (1997) also

report that P. syringae treatments were compatible with applications of the scald-

inhibiting antioxidant DPA, the most important post-harvest treatment for refrigerated

apples stored under ambient atmosphere.

Improvement of P. syringae control was possible by combining different

techniques. Treatment with 4 mg mL" 2-deoxy-D-glucose (DOG) was found to improve

P. syringae control of P. expansum on apples, providing nearly complete control with as

little 106 CFU P. syringae mL' (Janisiewicz 1994). Janisiewicz conjectures that DOG

inhibits P. expansum growth and conidial germination because the fungi internalize and

phosphorylate DOG, and the toxic metabolite produced by DOG phosporylation

accumulates to lethal concentrations. This theory is backed by previous work (Heredia

83

and Sols 1964). As mentioned above (El-Ghaouth and others 2001), 0.2% DOG has also

Q 1

been reported to improve C. saitoana (10 CFU mL" ) control of P. expansion in apples.

Interestingly, some researchers found that treatment with DOG numerically,

though not significantly, increased patulin concentration in apples inoculated with P.

expansion (Kazi and others 1997). More recently, members of this group reported that

treatment with DOG does significantly increase patulin production by P. expansum

(Paterson 2007).

While it is possible to control storage pathogens using P. syringae, most

researchers have found that the antagonist needed to be applied prior to challenge of the

fruit with P. expansum or other fungal pathogens. Latent pathogens were best controlled

when biological control was combined with a phytosanitation step, such as the previously

mentioned heat treatment (Leverentz and others 2000).

Lactobacillus spp.

Numerous species of lactic acid bacteria (LAB), many belonging to the genera

Lactobacillus, have been described as having in vitro antifungal and antimycotoxigenic

properties (Gourama 1997; Florianowicz 2001; Schniirer and Magnusson 2005). Some

authors (Schniirer and Magnusson 2005) have reported antifungal activity inplanta, and

in baked goods.. The mechanism of activity appears to involve competition for space and

nutrients (Schillinger and others 1996). Lactobacilli produce a class (Ha) of bacteriocins

specifically inhibitory to Listeria species (Ennahar and others 1999), numerous organic

acids, the broadly antimicrobial compound reuterin, antifungal fatty acids, numerous

84

antifungal low molecular weight compounds, phenyllactic acid, and antifungal cyclic

dipeptides (Schniirer and Magnusson 2005).

Although no studies specifically using apple have been conducted, there is reason

for optimism regarding the use of LAB to control fungal pathogens on fruit and in juice.

Effectiveness has been shown both in vitro and in various similar food systems, and there

' is widespread public acceptance of and familiarity with LAB, since they have a long

history of safe use. Recent work by Trias and others (2008) indicates that LAB may

protect apples from growth of the foodborne bacterial pathogens Listeria monocytogenes

and Salmonella typhimurium. However, LAB may act as spoilage organisms in apple

cider (Martinez-Viedma and others 2008).

Commercial availability of post-harvest biological control

products

Currently, P. syringae is the only post-harvest biological control agent currently

available in the U.S. for the reduction of storage rots on produce. Bio-Save® 10 and 11

(Jet-Harvest Solutions, Apopka, FL) are formulations of proprietary strains of P.

syringae, meant for use as post-harvest biological control agents (JetHarvest 2008). They

are both EPA registered pesticides (EPA 2000b) and Washington State Department of

Agriculture registered organic materials (WSDA 2009), and BS-10 is OMRI listed

(OMRI 2009).

Some strains of P. syringae have been found to be virulent pathogens of plants

(Guttman and others 2002). However, the strains of P. syringae included in the Bio-

85

Save® formulas are saprophytic, and pose no threat to intended fruits or plants that

produce them (Stockwell and Stack 2007); small lesions have been observed in Persian

o i

lime, when fruit was inoculated with 10 CFU raL" P. syringae (Smilanick and others

1996).

Previously, other products have been available, and others are in the marketing or

developmental stages.

The yeast Candida oleophila, enjoyed limited success as the product Aspire™,

marketing for controlling post-harvest Penicillium rots on pome fruits (Droby and others

2003) and citrus (Droby and others 1998; Bar-Shimon and others 2004).

Cryptococcus albidus, found to control post-harvest rots caused by P. expansion

and other fungi on apples (Fan and Tian 2001; Chan and Tian 2005), was packaged as

Yield Plus™ in South Africa (Chincholkar and Mukerji 2007).

Serenade™ (AgraQuest USA) was a commercially available post-harvest

biological control product based on the bacteria Bacillus subtilis, and indicated for the

control of pre- and post-harvest storage rots of many crops, including P. expansum on

apples (Marrone 2002).

Metchnikowia fructicola has been developed into a product, Shemer™, which is

being marketed in the Middle-East for post-harvest control of Botrytis cinerea on pepper

and tomatoes, and control of Rhizopus stolonifer on sweet potatoes and peaches

(Blachinsky and others 2007).

Neova Technologies (Abbotsford, British Columbia, Canada) is working on

commercializing a mixture of Candida saitoana and either chitosan or lysozyme,

marketed to control P. expansum on apples and citrus (Droby and others 2009).

86

EXPERIMENTAL OBJECTIVES

Numerous options are available to apple growers and cider producers wishing to

reduce the incidence of patulin in their product. Current practices that reduce patulin in

apple cider include washing fruit with a dilute chlorine solution, storing fruit and cider

under refrigeration, culling rotten fruit and excising rotten portions of fruit, properly

phytosanitizing the orchard, and applying synthetic fungicides to apples post-harvest.

While these measures all focus on reducing populations of patulin-producing fungi, the

only option currently available that offers both curative and protective efficacy is post-

harvest application of a fungicide with some degree of persistence. Most other methods

are fungistatic and prevent spread or development of infestation, while synthetic chemical

fungicides tend to be both fungistatic and fungicidal. In practice, the goal is always to

prevent rather than cure disease.

As discussed above, these commonly employed techniques can potentially reduce

the final patulin levels in apple cider, but all have shortcomings in one or more areas,

including effectiveness, cost, and environmental or worker health concerns. Less

commonly employed, experimental techniques focus on removing patulin after it has

been formed, preventing mycotoxin synthesis, or preventing fungal growth.

Removal of patulin may be suitable for producers of apple juice, where clarity is

more commercially relevant than retention of particulates and volatiles. However, it is

precisely these particulates (pulp) and volatiles that distinguish cider (or "cloudy juice",

as it is sometimes referred to in Europe) from juice. Filtration or chelation of apple cider

would inevitably lead to a decrease in cider quality, and would potentially lead to

87

customer refusal of the product. Biotransformation may reveal itself to be a workable

method of reducing extant patulin levels in contaminated juice, but currently seems to

work best in fermented products, such as hard cider or cider vinegar.

As previously mentioned, prevention of the formation of patulin offers a number

of obvious advantages over the removal of pre-formed toxin. Patulin may be reduced by

either preventing patulin-producing fungi from producing the mycotoxin, or by

preventing patulin-producing fungi from growing and reproducing at all. While methods

of preventing the formation of patulin by extant fungi may be included as part of a patulin

reduction strategy, the prevention of fungal growth and reproduction is a more

comprehensive, prophylactic, and effective strategy.

The prevention of mold growth on fruit is a season-long endeavor that includes

tree care, orchard sanitation, integrated pest management strategies to reduce incidence

and severity of fungal diseases in the field (for instance, application of fungicides during

peak fungal growth or reproduction, prevention of insect damage to fruit), and a harvest

date that optimizes apple maturity. However, without proper post-harvest handling and

treatments, all pre-harvest methods of mold reduction could be rendered moot. For

example, even if apples are brought in from the orchard sound and sanitary they could

still become damaged and/or infected with patulin-producing fungi. Handling fruit

roughly, storing fruit or juice at ambient temperatures (deck storage), or storing fruit or

juice in improperly sanitized equipment could all facilitate fungal infestation and lead to

patulin accumulation within fruit or juice.

Post-harvest measures to reduce fungal growth include keeping fruit cold prior to

pressing and immediately chilling pressed cider. The Maine Department of Agriculture,

88

Food, and Rural Resources already mandates proper storage temperatures of juice

(although published work suggests that storage temperatures should be lower for

maximum patulin reduction), and most or all producers recognize this step as critical to

ensure both quality and safety of apple cider. Pasteurizing and preserving juice with

chemical additives may reduce microbial populations, but neither offers complete control

of patulin-producing fungi. Furthermore, within the State of Maine, there is a

considerable market for cider that is not pasteurized and/or has no preservatives added,

and while this demand exists, cider producers will continue to offer these products. The

goal of this project was to develop techniques that would be affordable and appeal to

cider producers at all levels who desired to adopt strategies in reducing patulin levels in

their cider products.

Along with current measures, disinfecting apple surfaces (curing) and keeping

them clean (protecting) prior to pressing seems to be the most viable and comprehensive

approach for preventing post-harvest growth and reproduction of patulin-producing fungi

on fruit and in juice, and, ultimately, for reducing patulin in apple cider. It is essential to

both cure and protect apples against fungal growth to reliably and consistently reduce

patulin in cider.

CURING

Post-harvest treatments with AA and PAA have been found to cure microbial

infestations (sanitize the surface of the fruit), and are effective against a broad range of

microbes, including bacterial and fungal storage plant pathogens, as well as human

89

pathogens. Application with fumigation is more desirable than with drenches or dips, as

dip tanks can act as a medium for cross-contamination and effectively inoculate all fruit

with pathogens. Fumigation uses less product, usually dissipates from fruit surfaces

without leaving a residue, reaches all heights and orientations within a storage room, and

diffuses through spaces to penetrate protected areas of fruit not accessed by dips or

drenches. When using acids, fumigations tend to be more effective than treatment with

liquid-phase acid, due to the prevalence of undissociated acid in the vapor-phase.

Post-harvest treatments of fruit with vapor-phase AA have been shown to reduce

fungal decay in various fruits, but using vapor-phase vinegar (5% acetic acid) to reduce

P. expansum has been minimally reported. Elaborate fumigation mechanisms were

constructed or construed for these trials, and in some cases, the cost was considerable.

Interesting work involving aerosolized disinfectants suggested an alternative.

Using a "patented, proprietary process involving electrochemical means," Oh and

others (2005) aerosolized PAA to disinfect a semi-trailer, and reported that small droplet

size (<2 jum) was necessary to form a fog that would remain suspended, and thus reach all

portions of the chamber. In their concluding remarks, the authors suggest that new home

humidifiers may be capable of producing such small droplets, and therefore may provide

a resource for fumigation operations.

To our knowledge, no research has reported on the use of a home humidifier to

fumigate crops post-harvest. The home humidifier has appeal for a number of reasons,

including low cost, small size, and availability. A home humidifier may be a suitable

surrogate for a more expensive, larger, specialty fumigation unit, and may provide

90

sufficient coverage for the relatively small operations that make up most cider production

in the State of Maine.

Preliminary trials indicated that some commonly available, less-expensive ($30-

50), cold-mist humidifiers (including ultrasonic models capable of producing relatively

small droplets) did not provide adequate treatment. A hot steam vaporizer was trialed

and found to provide superior coverage. Since heat vaporizes (as opposed to

aerosolizing) the acid, it is reasonable to suspect that more undissociated acid is delivered

by vaporization than would presumably be delivered by aerosohzation, in which the acid

is in the liquid phase.

Vinegar vapor has only been investigated as a sole treatment, and not as part of a

combination of treatments for curing and protecting against P. expansum infestation of

apples.

At the time of writing, only limited research had been published on the use of

PAA to reduce fungi on apples, and PAA had not been assayed for antifungal activity

against Penicillium spp. on any produce.

PROTECTING

Aside from using post-harvest fungicides, protecting fruit from fungal infestation

is generally accomplished by storing fruit under refrigeration (either in ambient air or a

controlled atmosphere), treating fruit with ripening retarders such as 1 -

methylcyclopropene (MCP), or treating fruit with biological control agents (BCA). The

use of post-harvest applications of microbial BCA to protect produce from fungal

91

diseases in storage has been widely documented, but not widely adopted. As mentioned

above, BCA generally lose efficacy as latent mold counts rise. Therefore, BCA require a

pre-treatment to reduce latent fungi for optimal efficacy. While many antagonists have

been isolated and tested, only one post-harvest biocontrol product for the reduction of

fungal pathogens on apples and other fruit is commercially available. Rather than

looking to find another antagonistic isolate, we chose to look at the use of a commercially

available (but not widely used) product, in combination with the application of vinegar

vapor by a novel delivery system. Combinations of BCA and various curative treatments

have been researched; however, combining BCA with vinegar vapor or PAA vapor has

not been reported on.

OVERALL OBJECTIVES

A series of experiments were designed to assess potential combinations of

established techniques to cure infestations and to protect apples from future infestations

of patulin-producing molds. The goal of these experiments was to establish an

inexpensive, effective, relatively easy to implement, sustainable technique to both cure

and protect harvested apples from any infestation of molds that produce patulin. The

experiments were intended to verify the curative properties against latent molds and P.

expansum of white vinegar (WV) vapor and peracetic acid (PAA) vapor, to compare

efficacy of WV vapor (Hannaford Bros. Co., Scarborough, ME, U.S.A.) to the vapor of a

commercially available PAA product (Jet-Oxide™, Jet-Harvest Solutions, Longwood,

FL, U.S.A.), to verify protective efficacy of a commercially available BCA product (Bio-

92

Save®, Jet-Harvest Solutions, Longwood, FL, U.S.A.) against further mold infestation,

and to assess whether combining either fumigation with BCA improved the control of

latent fungi or inoculated P. expansum. The aim was to utilize literature-supported mold

reduction techniques in a novel combination, while investigating a novel fumigation

method for the delivery of AA and PAA vapor.

93

MATERIALS & METHODS

Fresh, hand-picked Mcintosh apples were obtained from Highmoor Farm, in

Monmouth, Maine during September of 2008. Apples were stored under ambient

atmosphere, at refrigerated temperatures (39-41 °F), until ready for use. All apples were

processed within 6 weeks.

Apples were removed from the refrigerator, allowed to reach room temperature,

and then processed. Treatments consisted of various combinations of fumigation, with

either distilled WV (Hannaford Bros. Co., Scarborough, ME, U.S.A.) or a PAA solution

containing 0.2% Jet-Oxide™ (Jet-Harvest Solutions, Longwood, FL, U.S.A.); application

of Pseudomonas syhngae biocontrol agent (Bio-Save® 10 or 11, Jet-Harvest Solutions,

Longwood, FL, U.S.A.); and inoculation with Penicillium expansum (Figures 1-3).

Experiments were designed to control for any potential protective properties of WV

vapor; to verify that it is actually the WV vapor, and not the process of fumigation, that is

curative; and to illuminate any potential curative properties of the BCA treatment.

The work of Sholberg and others (Sholberg and Gaunce 1995; Sholberg 1998;

Sholberg and others 2000) provided a working method for the fumigations. The delivery

system and duration of fumigations were a novel application, based on concluding

remarks by Oh and others (2005).

94

Figure 1. Summ

ary of treatments in T

rial One.

95

B C D

T T

G H

T T T T

Treat with BCA

Fumigate with WV

noculate with

• '. expansurk

I Inoculate

with '. expansu

Inoculate with

'. expansun\

Fumigate with WV

Hold at high

:emperature

Fumigate with WV

Wound

Figure 3. Sum

mary of treatm

ents in Trial T

hree.

97

r

Fumigations were carried out in a walk-in cooler with a volume of 19.2 m3. The

cooling unit was turned off, and the room was brought to ambient room temperature.

With the apples in the room, fumigant was loaded into a warm mist humidifier (Vicks®

model V745, Proctor & Gamble, Cincinnati, OH, U.S.A.) set on "High", and a small fan

(Honeywell®, Morristown, NJ, U.S.A.) was vertically oriented and placed in the middle

of the room, set on "High". The door to the cooler was then closed, and the humidifier

allowed to completely vaporize the fumigant for approximately 20 h.

P. expansum cultures were provided by Dr. David Lambert of the University of

Maine Department of Plant, Soil, and Environmental Sciences. They were isolated from

local apples, and then maintained on Potato Dextrose Agar plates at ambient room

temperature throughout the study. Patulin production capability of this strain of P.

expansum was verified by HPLC-MS-MS during the spring of 2009, using an HPLC

method developed in the lab of Dr. L. Brian Perkins, and then applying a modified

version of the MS-MS method described by Sewram and others (2000) described in

Appendix A. Samples of apple tissue that had been inoculated with P. expansum and on

which visible blue mold developed were then analyzed and found to contain high levels

of patulin. This affirmed that a patulin-producing strain of P. expansum was used in

these trials.

To prepare the P. expansum inoculum, we followed a method described by Dr.

Seanna Annis of the University of Maine School of Biology and Ecology, in a personal

communication (Annis 2008). Spores were washed off the PDA plate with a solution of

0.01% Tween 20 in de-ionized H2O (dl H2O), filtered through sterile cheesecloth into a

sterile 100 mL beaker, and then adjusted to a concentration of 10 CFU mL"1, using a

98

hemacytometer to determine proper dilution ratios. Aliquots of this solution were frozen

in glass cryovials, and then thawed and diluted to experimental concentration in

preparation to be used in each trial.

P. expansum inoculum density was verified by plating and counting 0.1 mL serial

dilutions of the 106 CFU ml"1 solution on Yeast and Mold Petrifilm™ (3M™, St. Paul,

MN, U.S.A.), and counting colonies after a three-day incubation at 20 °C. Yeast and

Mold (YM) counts for the verification tests were between 2.0-2.6 x 106 CFU mL"1.

After all treatments associated with the treatment group were performed, a sterile

blunt-tipped glass rod was used to create four 2-3 mm deep wounds, measuring 4-6 mm

across, encircling the calyx end of each apple. The glass rod was sterilized between each

apple by dipping the rod in 95% ethanol, passing it through flame, and allowing it to

briefly cool before wounding the next apple. After all apples were treated, they were

stored in covered plastic bins, at ambient room temperature, under a laminar flow hood,

until further analysis after 2-3 weeks. Three individual trials were conducted, to verify

results, to attempt to control for potentially confounding variables that arose, and to

compare different concentrations of fumigant.

Following storage, apple wounds were visually inspected for disease, and sampled

for microbiological analysis. Sterile, rayon-tipped swabs, containing letheen neutralizing

buffer (Quick Swab, 3M™, St. Paul, MN, U.S.A.) were utilized to swab apple wounds,

following incubation. The swab was then shaken vigorously in the buffer solution, to

release any microbes into the liquid; the buffered liquid from one swab-tube was

therefore a full strength sample of microbiota from all four wounds on a single apple.

This 1.0 mL liquid sample was then serially diluted in buffered H2O (1% peptone) and

99

plated on 3M™ Petrifilm™ Yeast & Mold (YM) Count Plates (St. Paul, MN, U.S.A.),

which were incubated at 20 °C for three days before being counted.

As each swab represented a sample of four wounds, the calculated YM count

(observed count multiplied by the appropriate dilution factor) for each apple was divided

by four, to obtain the mean YM count for the wounds on a single apple (see Tables B1 -

B3 in Appendix B for data). This number was used as the raw count for all statistical

analyses. In some cases, the YM film representing the most dilute solution was

uncountable, due to a very high concentration of colonies. In thes cases of there being

too many colonies to count on a plate, these plates were considered to contain 300

colonies, and the YM count was then calculated by multiplying 300 x the dilution factor

for that group.

TRIAL ONE

For the first trial, apples were divided into ten treatment groups (Figure 1), each

containing 10 apples except for the control group (Group A), which contained 5 apples.

Groups A, B, C1, and C2 investigated the effects and differences between fumigation

with PAA and WV, as well as the effect of the Pseudomonas syringae biocontrol agent

(BCA) on survival of latent populations of molds in apple wounds. Groups D, E, Fl, and

F2 investigated the effects and differences of fumigation with PAA and WV, as well as

the effect of the Pseudomonas syringae BCA on survival of artificially high populations

of the known patulin producer P. expansum, inoculated at a concentration of 1.0 x 103

CFU ml/1. Groups Gl and G2 investigated whether the application of BCA after

100

fumigation with PAA or WV, respectively, improved control provided by either

fumigation.

Group A was a control group, consisting of 5 apples which were simply wounded

and then stored. The other nine groups each consisted often apples.

Group B was treated with a BCA solution (Bio-Save® 10 LP, Jet-Harvest

Solutions, Longwood, FL, U.S.A.), prepared according to package directions, by

dissolving 14.0 g of the powder into 4.0 L dl H20. Apples were submerged in the

suspension for 15 minutes, and intermittently agitated gently with a steel spoon.

Group CI was treated by exposing the apples to 1.0 L of a vaporized PAA

solution, prepared by dissolving 2.0 mL Jet-Oxide™ (Jet-Harvest Solutions, Longwood,

FL, U.S.A.) into 1.0 L dl H2O. Once prepared, the fumigant was loaded into the warm

mist humidifier, and fumigated in the walk-in cooler as described above. Upon starting

the fumigation, the cooler was at 22 °C; after two hours, the temperature had risen to 25

°C; the next morning, after a 19 hour fumigation time, the room had reached 32 °C. This

high temperature was not anticipated, and may have presented a confounding variable.

Controls were taken to reduce this added variability both in this trial, and in subsequent

trials, as explained below.

Group C2 was treated with 1.0 L of vaporized 5% acidity distilled WV

(Hannaford Bros. Co., Scarborough, ME, U.S.A.) following the same procedures as for

group CI. The fumigation time was approximately 18 hours and the temperature of the

room again reached 32 °C before completion.

Group D was treated by submerging 10 apples into 20.0 L of dl H2O which

-1 1

contained 1.0 x 10J CFU mL"' P. expansum. This solution was prepared by thawing 20.0

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mL of solution containing 1 x 106 CFU mL"1 P. expansum, then dissolving this into 20.0

L dl H2O. The apples were submerged in a plastic bin containing the P. expansum

solution, agitated, and soaked for 15 minutes.

Group E was treated with the Pseudomonas syringae solution, as described above

for group B, then the Pseudomonas syringae solution and apples were added to the

Penicillium expansum solution, and the apples were soaked for 15 minutes, as described

above for group D.

Group Fl was treated with the Penicillium expansum solution, as described above

for group B, then fumigated with the PAA solution, as for group C1.

Group F2 was treated with the Penicillium expansum solution, as described above

for group B, then fumigated with the WV solution, as for group C2.

Group Gl was treated with the Penicillium expansum solution, and then

fumigated with the PAA solution, as described for group F1, before being treated with the

Pseudomonas syringae solution, as described for group B.

Group G2 was treated with the Penicillium expansum solution, and then

fumigated with the WV solution, as described for group F2, before being treated with the

Pseudomonas syringae solution, as described for group B.

As mentioned above, the groups that received a fumigation treatment also were

exposed to increasing temperatures through the course of the fumigation, ending at a

notably higher temperature. Others (Leverentz and others 2000; Leverentz and others

2003) have found that holding apples at 38 °C for four days was found to significantly

reduce the population of molds on the surface of the apples.

102

Therefore, the elevated temperatures to which the fumigated apples were exposed

may constitute a confounding variable, and it would be difficult to determine if an

observed difference in the survival of molds was due to the fumigation or due to the heat

exposure. Therefore, after all the other treatments, those apples in groups whose

treatments did not include a fumigation step were placed in the walk-in cooler overnight

to expose them to a similar level of heat (38 °C)that the fumigated apples received. The

experimental design of trial two took this potentially confounding variable into

consideration and included control measures.

TRIAL TWO

The second trial consisted often treatment groups (Figure 2), each with ten

apples. This trial was designed to replicate certain aspects of the first trial, while also

aiming to better control for the previously mentioned confounding variable presented by

the increased temperature that accompanied the previous fumigations. In order to

provide more control, in place of a PAA fumigation, a "Heat" treatment was included.

This heat treatment consisted of placing apples in the closed walk-in cooler, and allowing

the room to warm up over a period that was equal to the fumigation time of the other

treatments.

The key difference between this heat treatment and the heat treatment applied to

un-fumigated apples in the first trial was the order of operations. In this second trial, the

heat treatment was applied sequentially when a fumigation would have been

administered, namely, prior to treating apples with the Pseudomonas syringae BCA and

103

prior to wounding the apples. As mentioned above, researchers (Leverentz and others

2000; Leverentz and others 2003) have surmised that, in addition to the eradicative

properties of the heat itself, the observed increase in storage quality of and reduction of

molds on apples treated with heat was due to an increased rate of healing of small

wounds. Therefore, heating fruit prior to wounding was critical to ascertain direct effects

on fungi, rather than induced plant defense responses.

Groups A through D investigated the effects of Pseudomonas syringae biocontrol,

WV fumigation, and heat treatment on the extant population of molds on the surface of

apples. Groups E through H investigated the effects of Pseudomonas syringae

biocontrol, WV fumigation, and the heat treatment on the artificially inoculated

population of Penicillium expansum molds on the surface of apples, inoculated at a

concentration of 1.0 x 10 CFU mL" .

Group A of the second trial was a control group. Apples were simply wounded

and then stored before visual and microbiological analyses.

Group B was treated with the Pseudomonas syringae BCA (Bio-Save® 10, Jet-

Harvest Solutions, Longwood, FL, U.S.A.), prepared according to package directions, by

dissolving 14.0 g of the powder into 4000 mL dl H20. Apples were submerged in the

suspension, gently agitated with a steel spoon, and soaked for 15 minutes.

Group C was treated by exposing the apples to the vapor yielded from 1.0 L of

5% acidity, distilled WV (Hannaford Bros. Co., Scarborough, ME, U.S.A.), prepared by

loading the WV into the warm mist humidifier, and fumigating the walk-in cooler as

previously mentioned. Upon starting the fumigation, the cooler was at 22 °C; the next

morning, after a 20 hour fumigation time, the room had reached 31 °C.

104

Group D was treated by placing the apples into the walk-in cooler at 20 °C,

closing the door, and allowing the apples to remain in the cooler for 20 hours. Upon

removing the apples from the cooler, the cooler had reached 32 °C.

Group E was treated by submerging the apples into a suspension of 1.0 x 103 CFU

mL"1 Penicillium expansum, prepared by thawing and then dissolving 20.0 mL of a 1.0 x

106 CFU mL"1 solution into 20.0 L dI-H20. The apples were submerged in the solution,

agitated, and soaked for 15 minutes.

Group F was treated with the Pseudomonas syringae BCA as described for group

B, followed by inoculation with Penicillium expansum, as described for group E. As in

trial one, the suspension of Pseudomonas syringae BCA was added to the suspension of

Penicillium expansum inoculum before the final 15 minute soaking.

Group G was inoculated with Penicillium expansum, as described for group E,

and then fumigated with WV, as described for group C.

Group H was inoculated with Penicillium expansum, as described for group E,

and then treated with heat, as described for group D.

Group I was inoculated with Penicillium expansum, as described for group E,

fumigated with WV, as described for group C, and treated with the Pseudomonas

syringae BCA, as described for group B.

Group J was inoculated with Penicillium expansum, as described for group E,

treated with heat, as described for group D, and treated with the Pseudomonas syringae

BCA, as described for group B.

105

TRIAL THREE

The results from trial two indicated that the heat treatment reduced the population

of molds on apple surface versus molds on apples in the control groups with marginal

significance. However, fumigated apples harbored significantly fewer molds than the

heat treated apples, indicating that effects of the vapor fumigation were greater and more

relevant than those effects of heat treatment. For these reasons, the heat received by the

apples that were fumigated was determined not to be a significant confounding variable,

although it may constitute an additive factor in the effectiveness at fungal reduction.

Therefore, trial three aimed to replicate trial one, with the addition of a second

inoculation of Penicillium expansum for the last two treatment groups (Figure 3). The

change of treatment for these last two groups was intended to elucidate whether either of

the two fumigations improved the efficacy of the Pseudomonas syringae BCA treatment

to prevent further infestation, when challenged with a second inoculation.

Additionally, the fumigations were slightly altered. Since the WV treatment had

been highly effective, but had led to some damage to fruit and oxidation of copper piping

in the cooler, the volume of WV was reduced by 50% (from 1.0 L to 0.5 L). The volume

of PAA was reduced by 50% (from 1.0 L to 0.5 L), while concentration was increased by

100% (from 0.2% Jet-Oxide™ to 0.4% Jet-Oxide™), to maintain consistency with the

reduction in volume of the WV, while still providing the label-recommended

concentration of PAA recommended by Jet-Harvest (Jet-Harvest Solutions, Longwood,

FL, U.S.A.).

106

The third trial consisted often different treatment groups, but at this point the

supply of fresh, untreated apples that were still healthy was diminishing, so only seven

apples were included in each treatment group.

Group A was the control group. Apples were simply wounded and then stored

before visual and microbiological analyses.

Group B was treated with the Pseudomonas syringae BCA treatment, as described

above.

Group C was fumigated with vapor of 0.5 L of 5% acidity WV (Hannaford Bros.

Co., Scarborough, ME, U.S.A.), by loading the WV into the warm mist humidifier and

fumigating as described above. The starting temperature was 21 °C, and after a 17 hour

fumigation, the final temperature was 30 °C.

Group D was fumigated with 0.5 L of a PAA solution containing 0.2% Jet-

Oxide™ (Jet-Harvest Solutions, Longwood, FL, U.S.A.) to provide vaporized PAA. The

solution was placed into the warm mist humidifier and fumigating as previously

described. The starting temperature was 17 °C, and after a 24 hour fumigation, the final

temperature reached 30 °C.

Group E was inoculated by submerging the apples into a solution of dl H2O

containing 1x10 CFU mL" Penicillium expansum agitating, and soaking for 15

minutes.

Group F was treated with the Pseudomonas syringae BCA treatment for 15

minutes, and then inoculated with the Penicillium expansum solution by combining the

two solutions and soaking again for 15 minutes.

107

Group G was inoculated with the Penicillium expansum solution by soaking for

15 minutes, and then fumigated with 0.5 L of 5% acidity WV, as described above.

Group H was inoculated with the Penicillium expansum solution by soaking for

15 minutes, then fumigated with 0.5 L of 0.4% Jet-Oxide™ (Jet-Harvest Solutions,

Longwood, FL, U.S.A.), as described above.

Group I was inoculated with the Penicillium expansum solution, fumigated with

WV, treated with the Pseudomonas syringae BCA solution, and then inoculated a second

time with the Penicillium expansum solution, combining the Pseudomonas syringae BCA

and the Penicillium expansum inoculation solution for this final soaking.

Group J was inoculated with the Penicillium expansum solution, fumigated with

PAA, treated with the Pseudomonas syringae BCA solution, and then inoculated a

second time with the Penicillium expansum solution, combining the Pseudomonas

syringae BCA and the Penicillium expansum inoculation solution for this final soaking.

STATISTICAL METHODS OF ANALYSIS

Using JMP® software (SAS, Cary, NC, U.S.A.), ANOVA was initially performed

on data from each trial, to determine if there were any significant differences between the

mean YM counts of treatment groups within each trial. F-values ranged from O.0001-

0.0007, indicating that each trial had generated at least some significant variance among

treatment groups. Experiments had been designed with "planned comparisons" in mind,

and it was therefore possible to use the paired Student's /-test to analyze data in pairs

while keeping comparisons orthogonal and not increasing the risk of a Type-I error.

108

Significance level was set at a = 0.05, above which results were determined to be

insignificant (although in certain cases marginal results may be reported as such, with an

accompanying p-value, if doing so improves understanding of the results). P-values

reported are the probability of obtaining a greater \t\ than that t generated by the paired t-

test.

Raw data from counts of colonies that grew up when wound samples were plated

on 3M™ Petrifilm™ Yeast & Mold (YM) Count Plates (St. Paul, MN, U.S.A.) were

highly variable within each treatment group. The high degree of variability observed is

likely an emergent property of ecology and physiology of microbes that inhabit apple

surfaces and infect wounds. Outliers were common throughout the data collected, and it

was not rare for most apples in a group to harbor similar numbers of CFU per wound,

with a single apple or small number of apples exhibiting much higher counts. When

analyzing raw data counts, in spite of the above mentioned low F values (indicating

significant differences among at least some of the means), R values were only 0.27-0.38

(indicating that the response variation was not modeled effectively by ANOVA).

As the high variability of raw data skewed the descriptive statistics for each

treatment group, differences among groups of treatments were therefore obscured, and it

was necessary to somehow transform the raw data to elucidate the actual character of the

treatment response of a group of apples. Following the guidance of Dr. Eric Gallandt

(Associate Professor of Weed Ecology and Management for the University of Maine

Department of Plant, Soil, and Environmental Sciences), and Dr. William Halteman

(Associate Professor of Mathematics for the University of Maine School of Ecology and

109

Environmental Science), it was decided that a powerful transformation was needed to

explore the effects of various treatments on the orders of magnitude of spore production.

The rank transformation of raw count data was chosen after consideration of logio

transformation, square-root transformation, and various other "Box-Cox"-type (power)

data transformations (see Tables C1-C3 in Appendix C for a summary of the effects of

various transformations). The RT-1 method outlined by Conover and Iman (1981) was

used, and raw data within each trial were ordered and ranked from smallest to largest

(smallest observation = 1, second smallest = 2, and so on), with ties being assigned a rank

equal to the mean rank of all observations with the same value. Using the RT-1

transformation approximately doubled the R values associated with the ANOVA model

(R2 = 0.57-0.75), and allowed for more realistic and accurate separation of means.

However, in some cases, the rank transformation seemed to obscure what was

actually happening by compressing certain regions of data, in which case valuable

inferences could be drawn from the observation of raw data.

110

RESULTS

TRIAL ONE

The first trial investigated effects of fumigation with PAA and WV on both latent

fungi and inoculated Penicillium expansum in apple wounds, as well as differences

between fumigation with PAA and WV. Trial one also investigated effects of the

Pseudomonas syringae biocontrol agent on both latent fungi and inoculated P. expansum,

as well as whether fumigating with either PAA or WV prior to treating with BCA

improved the efficacy of BCA treatment. Results are summarized in Figures 4 and 5.

Figure 4. Rank-transformed counts of yeast and mold colonies per wound on un-inoculated apples in Trial One.

100-

90 H

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60 o* £ SO MH

40

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20-

10-

B

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B C1

Treatment Regime

• — » — »

C2

(Treatments with the same letter above box plot are not significantly different) Treatment A: Untreated control apples Treatment B: Apples that were treated with the biocontrol agent Treatment CI: Apples that were fumigated with peracetic acid solution Treatment C2: Apples that were fumigated with white vinegar

111

Figure 5. Rank-transformed counts of yeast and mold colonies per wound on P. exparaw/w-inoculated apples in Trial One.

(Treatments with the same letter above box plot are not significantly different) Treatment D: P. expansum-'moc\x\atQ& apples Treatment E: Apples treated with biocontrol agent prior to challenge with P. expansum Treatment Fl: P. expansum-'mocvX&tedi apples that were then fumigated with peracetic acid solution Treatment F2: P. ex/>a«swm-inoculated apples that were then fumigated with white vinegar Treatment Gl: P. expansum-mocwldiXtd apples that were then fumigated with peracetic acid solution, and then treated with biocontrol agent Treatment G2: P. expansum-m.ocx\\dXe,d. apples that were then fumigated with white vinegar, and then treated with biocontrol agent

J

Treatment of apples with white vinegar (WV) vapor cured infestations of both

latent fungi and artificially inoculated P. expansum. Un-inoculated apples showed no

visible fungal decay, regardless of treatment regime (Treatments A, B, CI, C2). Wounds

on un-inoculated apples treated with WV vapor (Treatment C2) had significantly lower

(pO.OOl) ranked yeast and mold (YM) counts after storage than those from wounds on

112

apples which were not fumigated (Treatment A). None of the un-inoculated apples

treated with WV vapor harbored any recoverable fungi, whereas un-inoculated, un-

fumigated apples had YM counts of 5.0 x 10 - 3.3 x 10 per wound.

All (10 of 10) P. expansum-'moculated apples that were not fumigated (Treatment

D) were visibly decayed; 6 of 10 apples showed visible fungal decay on all 4 wounds,

and P. expansum was recovered from 10 of 10 apples. In contrast, when P. expansum-

inoculated apples were fumigated with WV (Treatment F2), 0 of 10 showed any visible

decay, and P. expansum was recovered from only 1 of 10 apples. Ranked YM counts

from wounds of WV-fumigated apples were significantly reduced (p<0.001) compared

with those from wounds of inoculated apples which were not fumigated.

PAA vapor was not effective at curing latent fungal infestations in apple wounds,

but did reduce Penicillium expansum in apple wounds. Un-inoculated apples treated with

PAA vapor (Treatment C1) had ranked YM counts similar to those from un-inoculated

apples that were not fumigated (Treatment A), and the two treatments were not

significantly different (p=0.3558). While PAA vapor was unable to reduce YM counts

on P. expansum-'moc\i\&\Q& apples to undetectable levels, P. expansum-'moculated apples

that were then fumigated with PAA (Treatment Fl) showed significantly reduced

(/K0.0001) ranked YM counts compared with inoculated apples that were not fumigated

(Treatment D).

WV vapor was more effective than PAA vapor at reducing mold populations in

apple wounds. Ranked YM counts from wounds of un-inoculated apples treated with

WV vapor (Treatment C2) were significantly lower (pO.OOOl) compared to un-

inoculated apples treated with PAA vapor (Treatment CI). Likewise, ranked YM counts

113

from wounds of P. expansum-inoculated apples treated with WV vapor (F2) were

significantly less (pO.OOOl) than those from P. expansum-'moculated apples treated with

PAA vapor (Fl).

The Pseudomonas syringae biocontrol agent (BCA) significantly reduced

(p=0.0079) YM counts from wounds of both un-inoculated and P. expansum-mocvXaied

apples. When applied to un-inoculated apples, the BCA treatment (Treatment B)

significantly reduced the ranked YM count after storage compared to control apples

(Treatment A). When applied to apples prior to inoculation with P. expansum, the BCA

protected apples from mold growth: ranked YM counts from wounds on P. expansum-

inoculated wounds that had been protected with BCA (Treatment E) were both

significantly lower (/K0.0001) compared to those from P. expansum inoculated apples

that were not treated with BCA (Treatment D) and were not significantly different

(p=0.3029) from ranked YM counts of wounds on un-inoculated control apples

(Treatment A).

Treating apples with the BCA did not improve control of P. expansum provided

by either PAA or WV vapor. Ranked YM counts from wounds on apples that had been

inoculated with P. expansum, treated with vapor of PAA, and treated with the BCA

(Treatment Gl) were essentially identical to those from apples that did not receive the

BCA treatment after being inoculated with P. expansum and fumigated with PAA vapor

(Treatment Fl) (p=0.5712). Similarly, ranked YM counts from wounds on apples that

had been inoculated with P. expansum, treated with vapor of WV, and treated with the

BCA (Treatment G2) were essentially identical to those from apples that did not receive

114

the BCA treatment after being inoculated with P. expansum and fumigated with WV

(Treatments F2) (p=0.2277).

TRIAL TWO

The second trial was designed to replicate certain aspects of the first trial, while

also looking into potential effects of the confounding variable presented by the elevated

temperature that accompanied both fumigations by including a "Heat'" treatment. As it

had shown less promise, the PAA fumigation was omitted from this trial, and only WV

vapor was investigated in this trial. Results are summarized in Figures 6 and 7.

Figure 6. Rank-transformed counts of yeast and mold colonies per wound on un-inoculated apples in Trial Two.

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B C Treatment Regime

D

(Treatments with the same letter above box plot are not significantly different) Treatment A: Untreated control apples Treatment B: Apples that were treated with the biocontrol agent Treatment C: Apples that were fumigated with white vinegar Treatment D: Apples held at T < 32 °C overnight ("heat treatment")

115

Figure 7. Rank-transformed counts of yeast and mold colonies per wound on P. expansum-moc\AdL\Qd apples in Trial Two.

100

90

80 H

70

60 H

A AB BCD

Treatment Regime

(Treatments with the same letter above box plot are not significantly different) Treatment E: P. expansum-mocxxXdlQd. apples Treatment F: Apples treated with biocontrol agent prior to challenge with P. expansum Treatment G: P. expansion-inoculated apples that were then fumigated with white vinegar Treatment H: P. ex/rara-wm-inoculated apples that were then held at T < 32 °C overnight ("heat treatment") Treatment I: P. expam'wm-inoculated apples that were then fumigated with white vinegar, and then treated with biocontrol agent Treatment J: P. expansum-moc\AdL\Q& apples that were then held at T < 32 °C overnight ("heat treatment"), and then treated with biocontrol age"* eent

WV vapor was again found to be effective at reducing both latent fungi and

inoculated P. expansum in apple wounds. Considering un-inoculated fruit treated with

WV vapor (Treatment C), 8 of 10 apples had no recoverable latent fungi in wounds; 1

apple had 99% lower, and 1 apple had 40% lower raw YM counts than the average raw

count from wounds in the un-treated control apples (Treatment A). When inoculated

116

with P. expansum prior to WV fumigation (Treatment G), 8 of 10 apples treated with WV

vapor again showed no recoverable fungi in wounds, and the remaining 2 apples had

>99.9999% lower YM counts than the mean count of un-fumi gated apples (Treatment E),

a 6 log io reduction. The difference between YM counts from apples treated with WV

vapor and those from untreated apples was significant (p<0.000\) for both un-inoculated

and P. ex/xmsw/w-inoculated apples.

The heat treatment was found to slightly reduce counts of both latent fungi and

inoculated P. expansum. When un-inoculated apples were exposed to the heat treatment

(Treatment D), the average raw YM count in wounds was reduced by 64% compared

with control apples (Treatment A). This equated to a significant reduction (p=0.02),

when YM counts were rank-transformed. When P. expansum inoculated apples were

treated with heat (Treatment H), 8 of 10 showed a marked decrease (>99%) in raw YM

counts when compared to apples that had been inoculated with P. expansum but not

treated with heat (Treatment E). When considering the group as a whole, the difference

in ranked YM counts between heated and unheated apples was significant (p=0.03), in

spite of very high counts on the final 2 heat-treated apples.

WV vapor was substantially more effective than the heat treatment at curing both

latent fungi and P. expansum. Ranked YM counts from wounds on un-inoculated apples

treated with WV vapor (Treatment C) were significantly fewer (p=0.0038) compared to

counts from wounds on un-inoculated apples treated with heat (Treatments D). Similarly,

ranked YM counts from P. expansum-mocvAaled. apples treated with WV vapor

(Treatment G) were significantly fewer (/K0.0001) than counts from wounds on P.

expansum-mocxAattd apples treated with heat (Treatment H).

117

In trial two, the Pseudomonas syringae BCA treatment was not effective at

reducing either latent fungi or inoculated Penicillium expansum. The difference between

ranked YM counts from wounds on un-inoculated BCA-treated apples (Treatment B) and

those from wounds on untreated control apples (Treatment A) was insignificant

(p=0.2131), although 8 of 10 apples treated with BCA showed markedly reduced raw

YM counts when compared with the average raw count from wounds on untreated apples

(73% fewer). The other two BCA-treated apples had YM counts that exceeded the

average of counts from untreated apples, indicating variable control. Similarly, as a

whole, ranked YM counts from the BCA treated, P. expansum-inoculated apples

(Treatment F) were not significantly different (p=0A924) from ranked YM counts from

untreated apples inoculated with P. expansum (Treatment E). However, when treated

with the BCA prior to inoculation with P. expansum, wounds on 7 of 10 apples had

>99% lower raw YM counts compared with the mean raw YM count from wounds on

inoculated apples that had not been treated with the BCA. The other three BCA-treated

apples had YM counts similar to those from untreated apples.

Again, control of P. expansum afforded by the WV vapor treatment was not found

to be improved by the inclusion of the BCA treatment after fumigation. YM counts from

wounds in apples that had been inoculated with P. expansum and then fumigated with

WV vapor (Treatment G) were essentially identical with YM counts from apples that had

been treated with the BCA after being inoculated and fumigated (Treatment I). In both

treatment regimes, 8 of 10 apples harbored no recoverable fungi, and counts from the two

treatment regimes were not significantly different (p=0.6827).

118

In contrast, control of P. expansum provided by treating apples with heat was

improved by including a BCA treatment of apples after the heat treatment. Ranked YM

counts from wounds on P. expansum inoculated apples that were treated with heat and

then BCA (Treatment J) were significantly less (p=0.0002) than ranked YM counts from

wounds on P. expansum-'mocxAsLtzd apples that were treated with heat only (Treatment

H).

Interestingly, treating inoculated apples with heat before applying the BCA

resulted in similar control of P. expansum compared to the control obtained by

fumigating fruit with vaporized WV prior to the BCA treatment. In the case of both

treatment regimes, 8 of 10 apples showed no recoverable fungi. The WV vapor was able

to completely control the onset of visible decay in all wounds on all apples, while the

heat treatment prevented decay in all but 2 apples, in which 1 of 4 and 3 of 4 wounds,

respectively, were visibly decayed by P. expansum. In those instances of visible decay,

fungal counts from wounds of apples treated with heat were more than an order of

magnitude greater than counts from wounds in WV vapor treated apples that harbored

recoverable fungi (7.5 x 10 conidia per wound compared with 2.6 x 10 conidia per

wound). However, when considered as a group, this difference was not significant

(p=0.5074), regardless of analysis or transformation of data.

TRIAL THREE

Results from trial two indicated that the heat treatment was responsible for some,

but not all, of the antifungal effects of the vapor treatments, and the vapor treatments

119

resulted in lower counts than the heat alone. Therefore, trial three was designed to

replicate trial one, investigating combinations of fumigations and BCA treatments, as

well as differences between fumigations with PAA and WV, and also to elucidate

whether either fumigation improved the protective capacity of the Pseudomonas syringae

BCA treatment. The liquid volume of WV used in the WV fumigation treatment was

halved, and the PAA concentration was doubled, while still reducing volume by one half,

to maintain equal fumigation volumes and still deliver the recommended amount of PAA.

Results are summarized in Figures 8 and 9.

Figure 8. Rank-transformed counts of yeast and mold colonies per wound on un-inoculated apples in Trial Three.

70-AB

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A B C D

Treatment

(Treatments with the same letter above box plot are not significantly different) Treatment A: Untreated control apples Treatment B: Apples that were treated with the biocontrol agent Treatment C: Apples that were fumigated with white vinegar Treatment D: Apples that were fumigated with peracetic acid solution

120

Figure 9. Rank-transformed counts of yeast and mold colonies per wound on P. expansum-moculated apples in Trial Three.

A

70-

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E F G H I J

Treatment ^

(Treatments with the same letter above box plot are not significantly different) Treatment E: P. expansum-moculated apples Treatment F: Apples treated with biocontrol agent prior to challenge with P. expansum Treatment G: P. expansum-moculated apples that were then fumigated with white vinegar Treatment H: P. expansum-moculated apples that were then fumigated with peracetic acid solution Treatment I: P. expansum-moculated apples that were then fumigated with white vinegar, then treated with biocontrol agent, and then challenged again with P. expansum Treatment J: P. expansum-moculated apples that were then fumigated with peracetic acid solution, then treated with biocontrol agent, and then challenged again with P. expansum

Fumigations in this trial were again highly effective. All wounds on un-

inoculated apples fumigated with either WV vapor or PAA vapor were free of visible

decay after storage. Similarly, no wounds in any of the 7 P. expansum inoculated apples

121

that were fumigated with WV vapor showed decay after storage; of 7 P. expansum

inoculated apples that were fumigated with PAA vapor, 1 apple had 1 wound with visible

decay.

The WV fumigation reduced latent fungi in apple wounds compared to un-

fumigated apples. Ranked YM counts from wounds on apples that were treated with WV

vapor (Treatment C) were significantly lower (pO.OOOl) than untreated control apples

(Treatment A). Treating un-inoculated apples with WV vapor reduced YM counts of

latent fungi to undetectable levels in all wounds on 6 of 7 apples; the remaining apple

harbored an average of 1 spore per wound. WV fumigation also significantly reduced

(p<0.000\) YM counts from wounds on P. expansurn-inoculated apples (Treatment G)

compared to YM counts from wounds on P. expansion-inoculated apples that were not

fumigated (Treatment E). Treating apples with WV vapor reduced YM counts from

wounds of P. expcmsum-inoculated apples to undetectable levels in all 7 apples.

PAA fumigation significantly reduced latent fungi in apple wounds compared to

un-fumigated apples. Ranked YM counts from un-inoculated apples that were fumigated

with PAA (Treatment D) were significantly lower (p=0.0009) than ranked YM counts

from un-treated control apples (Treatment A). PAA fumigation reduced YM counts to

undetectable levels in 3 of 7 un-inoculated apples, and the mean raw YM count from

wounds of PAA-fumigated apples was 96% less than the mean raw YM count from

wounds of un-fumigated apples.

Fumigation with PAA vapor also reduced YM counts from wounds on P.

expansum-inoculated apples, compared to un-fumigated apples. Ranked YM counts from

P. expansum-inoculated apples that were fumigated with PAA (Treatment H) were

122

significantly lower (p=0.0001) than ranked YM counts from P. exparaww-inoculated

apples that were not fumigated (Treatment E). PAA fumigation reduced YM counts from

wounds on P. expam'ww-inoculated apples to undetectable levels in 5 of 7 apples, and the

mean raw YM count from wounds of P. expansum inoculated apples was 87% less than

the mean raw YM count from wounds of P. ex/ra/Mww-inoculated apples that had not

been fumigated.

WV vapor fumigation reduced latent molds in apple wounds similarly to

fumigation with PAA vapor. The difference between ranked YM counts from wounds on

un-inoculated apples fumigated with WV (Treatment C) and those from wounds on un-

inoculated apples fumigated with PAA (Treatment D) was not significant (p=0.13).

When apples were inoculated with P. expansum, treating apples with WV vapor

(Treatment G) led to marginally lower ranked YM counts than those observed after

treating P. expansum-mocvXaied apples with PAA vapor (Treatment H); however, the

difference was not significant (p=0.0620).

The Pseudomonas syringae BCA provided a modicum of control for both latent

molds and P. expansum in apple wounds. Analyses of raw count data indicate that mean

raw YM counts from wounds on un-inoculated, BCA-treated apples (Treatment B) were

87% lower than the mean raw YM count from wounds on un-treated control apples

(Treatment A). However, ranked YM counts of latent conidia in wounds of BCA-treated

apples were not significantly different (P=0.1696) from ranked YM counts from wounds

on un-treated apples.

Similarly, raw count data indicates that the mean raw YM counts from wounds of

BCA-treated apples inoculated with P. expansum (Treatment F) were 99.9% lower than

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the mean raw YM counts from un-treated apples inoculated with P. expansum (Treatment

E). Ranked YM counts from wounds of BCA-treated apples inoculated with P.

expansum were significantly lower (/?=0.0481) than rank-transformed counts from

wounds in untreated apples inoculated with P. expansum.

Control capacity of the BCA treatment was improved by pre-treating apples with

WV vapor, but not by pre-treating apples with PAA vapor. When apples were pre-treated

with WV vapor before being treated with the BCA and inoculated with P. expansum

(Treatment I), 6 of 7 apples remained completely free of recoverable fungi during

storage, with 1 showing signs of decay in 2 of 4 wounds. None of the wounds on apples

treated with the BCA and then challenged with P. expansum (Treatment F) were

completely free of recoverable fungi after storage. The difference between ranked YM

counts from wounds on the apples pre-treated with WV vapor (Treatment I) and those

from wounds on apples that were only treated with BCA before (Treatment F) being

inoculated with P. expansum was significant (p=0.0031). Notably, ranked counts of

conidia from wounds on these apples that had been fumigated with WV before the BCA-

treatment and P. expansum inoculation (Treatment I) were also significantly lower

(pO.OOOl) than ranked YM counts from wounds on untreated (control) apples

(Treatment A), in spite of the former treatment regime including two separate

inoculations of P. expansum.

When apples were pre-treated with PAA vapor prior to treatment with the BCA

and inoculation with P. expansum (Treatment J), 2 of 7 apples remained completely free

of recoverable fungi during storage. The remaining apples were highly variable with

regard to recoverable fungi, with 2 showing signs of decay in 1 of 4 wounds each. As

124

mentioned above, none of the wounds on P. expansum-c\\a\\enge.&, BCA-treated apples

that had not been fumigated with PAA vapor (Treatment F) were completely free of

recoverable fungi after storage. The difference between ranked YM counts from wounds

on apples pre-treated with PAA vapor (Treatment J) and ranked YM counts from wounds

on apples that were only treated with the BCA before being inoculated with P. expansum

(Treatment F) was not significant (p=0.6311). Again, ranked YM counts from wounds

on these apples that had been fumigated with PAA before the BCA-treatment and P.

expansum inoculation (Treatment J) were similar to rank-transformed counts of conidia

from wounds on untreated (control) apples (Treatment A); the difference between counts

of recoverable molds from the two groups was not significant (p=0.0730).

125

DISCUSSION

SUITABILITY OF INOCULUM

In general, inoculum density is a major factor in whether disease will develop,

and without a sufficient quantity of pathogens, disease may not be observed after

inoculation. In these experiments, the inoculum density of P. expansum was similar to

concentrations of P. expansum found in flume and/or wash water of apple processing

operations in which measures are taken to control the spread of pathogens (such as

sorting and grading). Work by Spotts and others (1988) indicates that P. expansum

concentrations vary widely in dunk and dip water used in apple processing, but P.

expansum was recovered most frequently at mean concentrations of 1-2 logio CFU mL"1.

In later work by Spotts and Cervantes (1993), the authors reported that 44% of the

samples of dunk tank water from apple and pear processing operations in the Pacific

Northwest region of the U.S. contained concentrations of P. expansum between 1-2 logio

CFU mL" . Sydenham and others (1997) have reported mean P. expansum concentrations

of 3 logio CFU g"1 in unpasteurized juice that was pressed from sorted and washed apples,

which had been stored at ambient deck temperatures for 7 days. Taking the mean P.

expansum concentration in dunk tanks as between 1-2 logio CFU mL"1, and recognizing

that mean counts approached 3 logio CFU mL"1 in juice pressed from un-refrigerated

apples that had been stored for 7 days post-harvest, inoculation density utilized for P.

expansum challenges in the following experiments was 3 logio CFU mL"1.

126

Inoculations used were sufficient to induce onset of decay in all trials, though the

difference in ranked YM counts between inoculated and control apples was significant in

the first trial only (p=0.0209) (see Tables C1-C3 in Appendix C). This may have been

due to the proliferation of fungi during storage, on the surfaces of un-inoculated control

apples, or other unforeseen factors. Also, as recovered fungi were not positively

identified, the YM counts were only a generalized "snapshot" of the microbial population

on apple surfaces and in wounds, and therefore may have included non-pathogenic (or

even antagonistic) species.

Interestingly, YM counts from an apple wound were not always an accurate

predictor of whether fungal decay would develop. Many apples harbored 2.0 x 105 CFU

-4 .0x l0 5 CFU per wound but showed no visible decay, and one sound-looking apple

harbored 5.4 xlO5 CFU per wound. In other cases, decay set in without a high fungal

load; as few as 4.5 x 10' CFU per wound led to visible decay in one instance.

The implications of these findings are extremely relevant to cider producers and

apple growers: apples may appear sound and clean, while in reality being contaminated

with very high fungal loads. If these fungi include patulin-producers (which is likely

considering the ubiquity of Penicillia), it would be possible for a single apple that appears

sound - and thus, would not be culled or discarded based on visual appearance - to

contaminate a quantity of cider. It is for this reason that many producers who think that

they are taking adequate control measures to reduce the incidence of patulin in cider may,

in fact, generate cider that becomes contaminated with patulin during processing or

storage.

127

EFFECTS OF THE BIOLOGICAL CONTROL AGENT

The BCA was variably effective at reducing the proliferation of extant molds, and

slightly more consistent at protecting apples against a challenge with P. expansum. Trial

one was the only series of experiments in which the BCA treatment substantially reduced

the growth of latent fungi, as evidenced by significantly lower (p=0.0079) ranked YM

counts from un-inoculated apples analyzed after storage, when compared with apples that

were not treated with the BCA before storage. Nonetheless, consideration of raw data

generated by the other two trials provides valuable insight, and suggests at least some

control effects associated with the BCA treatment.

In trial two, 7 of 10 un-inoculated apples treated with BCA had raw YM counts

that were lower than the mean raw YM count from un-treated control apples; 1 apple had

YM counts similar to the mean raw YM count from un-treated apples, and 2 apples had

YM counts that were appreciably greater than the mean raw YM count from untreated

apples. These results indicate the variable control of fungal disease provided by the BCA

treatment, and also hint at the all-or-nothing ecology of fungal infection. If one spore

survives treatment, germinates, and grows out to reproduce, the fungal population

increases rapidly and exponentially, and counts of fungi from wounds of treated apples

will be similar to counts from wounds of untreated apples. When we exclude the three

outliers (cases in which something like the above-described phenomenon presumably

occurred), the mean ranked YM count from apples treated with BCA was significantly

lower (p=0.0226) than that from untreated control apples. Still, performance was not

128

consistent, and there is certainly a need for improved technique to satisfactorily control

crop disease with this post-harvest BCA treatment.

In trial three, raw YM counts from all 7 un-inoculated apples treated with BCA

were lower than the mean raw YM count from un-treated control apples; all 7 un-

inoculated apples treated with BCA had lower YM counts than 5 of 7 un-treated control

apples. Although not a statistically significant difference (p=0.\696), in trial three, the

mean raw YM count from apples treated with the BCA was almost an order of magnitude

lower than the mean raw YM count from untreated apples (the BCA provided a 1 logio

reduction of latent fungi over the duration of storage).

Although steps were taken to prevent confounding variability, this lack of

effective control may have been due to a number of reasons, including but not limited to

increased duration between harvest of apples and the treatments. During this time,

senescence, weakening of plant defenses, fungal growth and reproduction, or even

storage wounding may have confounded results. Although the BCA was vacuum sealed

and stored at -18° C after each use, it is possible that the BCA lost efficacy during

storage.

Compared to its ability to control extant fungi, the BCA was more consistent in its

ability to protect apples from fungal infection when apples were challenged with

pathogens after being treated with the BCA. In trials one and three, the BCA treatment

protected apples against a challenge with P. expansum, and ranked YM counts after

storage were significantly reduced (pO.OOOl in trial one and p=0.0481 in trial three) in

BCA treated apples when compared with un-treated, P. expansum-moculated apples. In

trial two, when ranked YM counts were analyzed, no significant difference (p=0.4924)

129

was observed between un-treated and BCA-treated apples challenged with P. expansum.

However, if we consider raw YM counts from trial two, treating apples with the BCA

prior to challenging with P. expansum significantly reduced (p=0.0361) YM counts after

storage, when compared to raw YM counts from apples that were not protected with the

BCA prior to being challenged with P. expansum.

These results fit our expectations regarding the potential of the BCA. Namely,

the BCA was quite effective at preventing onset of disease when apples protected with

the BCA were challenged with fungal pathogens, but the BCA was not highly effective at

reducing populations of established fungi. However, raw counts were generally variable,

and the BCA was neither able to provide complete control of latent fungi nor protect

apples completely against a challenge presented by an inoculum of P. expansum. The

BCA treatment appeared to inhibit the progression of fungal growth and/or

accompanying disease development, but was not capable of reducing populations

directly. These findings support the idea that, to maximize its protective effects, the BCA

should be combined with an eradicative step that is intended to reduce extant populations

of fungi.

EFFECTS OF FUMIGATIONS

Fumigating apples with WV vapor was a highly effective means of reducing

populations of both latent fungi and inoculated P. expansum, suggesting that this

technique may be suitable for inclusion in a plan to reduce patulin in apple cider. In

consideration of all three trials, when un-inoculated apples were treated with WV vapor,

130

latent yeasts and molds were reduced to undetectable levels in 24 of 27 apples, with the

remaining apples showing markedly and dramatically reduced YM counts. In all three

trials, treating un-inoculated apples with WV vapor significantly reduced (p<0.00\ in all

three trials) the mean ranked YM count when compared with the mean ranked YM count

from un-treated control apples, which indicates that WV vapor was effective at curing

latent infestations of yeasts and molds, which may include patulin-producing species of

fungi.

Similarly, when P. expansum-inoculated apples were fumigated, 24 of 27 apples

had no recoverable fungi; in all three trials, ranked YM counts from P. expansum-

inoculated apples that were treated with WV vapor were significantly lower (p<0.001 in

all three trials) than those from inoculated apples that were not treated with WV vapor.

These results suggest that, in addition to being broadly antifungal, treatment with WV

vapor significantly reduces populations of the known patulin producer, P. expansion, in

most cases to undetectable levels.

Fumigating apples with the commercially available preparation of PAA

consistently reduced populations of P. expansum, but was less effective at reducing

extant populations of yeasts and molds. In trial one and trial three (the two trials that

explored the use of PAA vapor), the mean ranked YM count from P. expansum-

inoculated apples treated with PAA vapor was significantly lower (p<0.0001 in both trial

one and trial three) than that from inoculated apples that were not treated with PAA

vapor. While the PAA treatment did not reduce yeast and mold populations on

inoculated apples to undetectable levels with the frequency that treatment with WV vapor

did (5 of 17 inoculated apples that were fumigated with PAA showed no recoverable

131

fungi; all five were from trial three), these results nonetheless indicate that treatment with

PAA vapor does dramatically reduce populations of the known-patulin producer, P.

expansum, and may be a suitable technique for preventing patulin formation in apples or

cider. While statistical comparisons between the trials (such as comparing results from

trial one to results from trial three) were not performed - due to differences in sample

sizes and confounding circumstantial variables - it is noteworthy that only in trial three,

when the volume of PAA was halved and the concentration doubled compared to trial

one, were yeast and mold populations reduced to undetectable levels in (5 of 7) P.

expansum-mocvXatedi apples treated with PAA vapor. All P. ex/?amwm-inoculated apples

that were fumigated with PAA in trial one (the lower concentration, higher volume

fumigation) harbored recoverable fungi, with YM counts as high as 2.1 x 103 CFU per

wound.

As mentioned above, the control of latent fungi provided by PAA vapor was more

variable than control of P. expansum. In trial one, un-inoculated apples treated with PAA

vapor harbored populations of latent fungi similar to those on un-fumigated apples:

ranked YM counts from un-inoculated apples that were fumigated with PAA were not

significantly different (p=0.3558) than counts from control apples, and raw counts for the

two groups were similar as well. In trial three, PAA vapor significantly reduced

(/?=0.0009) populations of latent fungi, compared to those found on un-fumigated control

apples, again suggesting the superiority of the fumigation formula used in trial three

compared to that used in trial one.

Of the two vapor treatments, the WV fumigations were more effective (and

impressive) than the PAA fumigations regarding ability to cure infestations of both latent

132

fungi and P. expansum. In trials one and three, the mean ranked YM count from un-

inoculated apples treated with WV vapor was lower than the mean ranked YM count

from un-inoculated apples treated with PAA vapor, although significantly different

(pO.OOOl) in trial one only. Data from trial three does, nonetheless, suggest a difference

in response between the two fumigation groups. Of 7 un-inoculated apples that were

fumigated with WV, 6 showed no recoverable fungi, with the final apple harboring an

average of 1.0 x 10° CFU per wound, for a mean raw YM count of 1.0 x 10" CFU per

wound for the WV vapor group. Contradistinctively, 4 of 7 un-inoculated apples

fumigated with PAA harbored recoverable fungi, of which 2 had YM counts on the order

oficrcFU per wound, giving a mean raw YM count of 6.0x10'CFU per wound for

the PAA vapor group. It is possible that higher concentrations of PAA would be more

effective at reducing latent fungi, but the concentration used in these experiments was the

recommended dosage listed on the label of the commercially available PAA solution.

Results were similar when using P. exparawm-inoculated apples; WV vapor was

more effective than PAA vapor at reducing populations of P. expansum, although

significantly so in trial one only. In trial one, the mean ranked YM count for P.

expansum-'m.ocx\\&\Qdi apples fumigated with WV was significantly lower (/K0.0001) than

the mean ranked YM count for P. exparaww-inoculated apples fumigated with PAA.

This statistical significance reflects the disparity between the WV vapor group, in which

9 of 10 apples harbored no recoverable fungi, and the PAA vapor group, in which all 10

apples harbored recoverable fungi, with 6 apples having 10-10 CFU per wound. As the

PAA fumigation was more effective in trial three, less difference was observed between

the YM counts from apples treated with WV vapor and YM counts from apples treated

133

with PAA vapor, and statistical analyses of the ranked YM counts revealed that this

difference was of marginal significance (p=0.0620). PAA vapor reduced P. expansum to

undetectable levels in 5 of 7 apples; however, the remaining 2 apples had counts on the

order of 105-106 CFU per wound, bringing the mean raw YM count to 2.2 x 104 CFU per

wound. WV vapor reduced P. expansum to undetectable levels in all 7 apples. Again, it

is possible that higher concentrations of PAA would be more effective at reducing P.

expansum, but the concentration used in these experiments was that recommended on the

label of the PAA solution.

Unquestionably, there exists a difference between the two treatment groups.

However, compressive effects of the rank-transformation obscure any differences

between these two relatively similar groups, and statistical analyses of raw data fare no

better at illuminating any discrepancy. This difference in response, even if minimal,

when combined with the numerous logistical advantages offered by using WV versus

using PAA (such as cost, availability, familiarity, potential to be produced by the user,

safety, and others), suggest that WV could at least stand in for PAA in operations using

fumigation for the reduction of fungal populations on the surface of apples. Furthermore,

even if the difference between the responses of the two fumigation groups is not

significant, the variability of the response is certainly different: PAA is less consistent

than WV vapor in its ability to cure infestations of molds.

However, the use of WV vapor comes with two caveats. First, WV vapor is fairly

corrosive. Exposed copper piping in the room used for these trials developed a thin layer

of verdigris oxidation, and it is reasonable to suspect that other exposed metal surfaces

may become corroded following exposure to WV vapor. While this oxidation was easily

134

removed with steel wool and detergent, it nonetheless poses a concern for anyone

interested in adopting this technique. Simple solutions may be offered by using a room

that has no exposed piping or wiring for the fumigation, outfitting an area with some sort

of vapor barrier to contain the fumigant for the duration of treatment, finishing the

treatment area with plastics or laminates, or using less fumigant to alleviate these effects.

Although the work represented here was concerned with finding a completely effective

dose, future work may elucidate a minimum inhibitory concentration of WV vapor that

does not damage exposed metal.

Second, WV vapor was mildly phytotoxic to apples in these trials. This result

was not unexpected; similar results have been reported by others using vaporized glacial

acetic acid (Sapers and others 2003), and Sholberg mentions the possibility of

phytotoxicity when using vinegar vapor (Sholberg and others 2000). Especially in trials

one and two, when the higher concentration of WV vapor was used, most fruit developed

lesions of damage around the calyx or at any small nick or cut in the skin. In trial three,

when the volume of WV was halved, these effects were minimized, but further work

would be needed in order to confidently determine a minimum inhibitory concentration

that did not damage fruit. Other varieties may be more susceptible to damage. It should

be noted that the damage was not extensive, and fruit fumigated with the quantity of WV

used in trial three would certainly be suitable for use in cider production, as cider apples

may have blemishes. Interestingly, WV fumigations were still effective at reducing YM

counts and preventing the onset of decay, even when treating apples that had been stored

for nearly 2 months.

135

EFFECTS OF COMBINING FUMIGATIONS AND BIOCONTROL

Although heavily researched and reported upon for over twenty years, the post-

harvest use of microbiological control agents for the alleviation of plant disease has not

caught on with growers, as evidenced by the number of products on the market today

(one), in light of how many have failed (at least five). Whether out of lack of familiarity

with these products, lack of product availability, or the more general cultural inertia that -

for better or worse - delays the adoption of new technology, incorporating beneficial

microbes into the arsenal of tools used in the management of plant disease has not been

widespread. As the greater public becomes familiar and comfortable with the use of

probiotics (in essence, microbiological control agents for the gut), as the internet

continues to facilitate the dissemination of information about new technology, and as

agriculture looks for solutions to the waning efficacy of current technology, biological

control - including the use of microbes as BCA - may very well gain more acceptance.

Major hurdles to widespread acceptance of biological control have traditionally

included the increased level of knowledge and technical expertise that is required for

successful use of biological versus physical or chemical control, and a lack of consistent

efficacy associated with biological control. However, these issues are more commonly

associated with pre-harvest field applications of BCA, such as entomological control (for

example, releasing predatory wasps to control insect pests or releasing mycophagous

mites to control fungal disease) or the use of microbes to control soil-borne diseases that

affect root crops or roots of crops (such as planting Allium species into straw inoculated

136

with beneficial microbes that antagonize white rot causative microbes, as described by

Avila Miranda and others (2006)).

The emerging field of post-harvest microbiological control is different, and

application is generally no more difficult, skilled, or complex than comparative chemical

control. Nonetheless, efficacy and consistency are still hurdles that need to be addressed,

and one such way of doing so involves combining a biological control agent, which is

meant to protect crops against future challenges, with an eradicative pre-treatment, which

reduces extant populations of plant pathogens. A major consideration and goal of these

experiments was to investigate the use of PAA or WV fumigation as an eradicative

treatment preceding application of a commercially available post-harvest BCA, meant to

protect produce, including apples.

When apples were inoculated with P. expansion, including a BCA treatment after

fumigation with either WV or PAA did not improve control of fungal growth during

storage provided by fumigation alone. In trial one, P. expam'wm-inoculated apples that

were fumigated with PAA and then treated with BCA harbored populations of fungi

similar (p=0.5712) to those on apples that had been fumigated with PAA only. BCA

treatment in this case actually led to insignificantly higher final YM counts, possibly by

providing a vector of cross-contamination in the drench-tank. This indicates that the

BCA alone may not be a sufficient substitute for chemical control to prevent the spread

of pathogens in wash water.

Similarly, in trials one and two, when P. expam'wm-inoculated apples were

fumigated with WV vapor and then treated with BCA, no improvement in control was

provided by the addition of the BCA treatment, and YM counts were similar for the two

137

treatment groups after storage (p=0.2277). However, it should be noted that improving

control provided by WV vapor was practically impossible; of 20 P. ex/?<ms«/w-inoculated

apples that were fumigated with WV in the first two trials, 17 apples harbored

undetectable levels of fungi.

Differences between the conditions of storage utilized in these experiments and

those in a commercial operation are also important in the understanding and

interpretation of these results. The storage times before analysis for apples in these trials

was 2-3 weeks, whereas apples may be stored for longer periods of time in commercial

growing and cider producing operations. Storage time of the apples is noteworthy

because of the intended effect of the BCA treatment, namely protection against further

infestation during storage, the likelihood of which may increase with an increase of

storage time.

Additionally, in these trials apples were surface sanitized (or at least disinfected),

placed into storage bins which had been sanitized, and then stored in a laminar flow

hood, providing numerous measures of protection against further infestation (hurdles)

during storage. In a commercial operation, this would not necessarily be the case

(although the potential for adaptation of aseptic technique to crop storage has obvious

implications), and an opportunity for infestation of fruit with fungal pathogens may be

present throughout the storage period, in the form of fungal load on storage equipment, in

the storage room, or even in the air. In these cases, any protective capacity would be

more greatly called upon, and presumably those apples treated with the BCA would be

less susceptible to fungal infestation than apples treated only with fumigant, which offers

no residual protection.

138

This discrepancy was addressed in the design of trial three, which included a

second P. expansum inoculation, meant to simulate an instance of the above-mentioned

challenge during storage. In the third trial, it was found that fumigating P. expansum-

inoculated apples with WV vapor prior to treating with BCA significantly improved the

capacity of the BCA to protect fruit against further infestation when challenged with P.

expansum (p=0.0036). This was expected, as the WV fumigation had been repeatedly

shown to reduce populations of both latent fungi and P. expansum, and numerous

publications (El-Neshawy and Wilson 1997; Leverentz and others 2000; Droby and

others 2003; Leverentz and others 2003; Spadaro and others 2004; Janisiewicz and others

2005) suggest that post-harvest biological control is most effective when combined with

an eradicative step.

Fumigating P. expajwwm-inoculated apples with PAA vapor prior to the BCA

treatment did not improve the capacity of BCA to protect fruit against a further

infestation when challenged with P. expansum. This result was most likely due to the

inability of PAA vapor to completely eradicate populations of P. expansum. Although 2

of 7 apples had no recoverable fungi, and 2 more apples had very low YM counts (1.9-

3.1 x 101 CFU per wound), the remaining 3 apples had high concentrations of/*

expansum (8.4 x 10 -1.3 x 10 CFU per wound), which were sporulating at the time of

analysis. Even if just one apple harbored visible growth of P. expansum (blue mold), this

would be enough to infect other apples in the group, or to overwhelm the BCA and

colonize apple wounds.

When comparing the two fumigations, we see that P. expansum-'moculated apples

fumigated with WV vapor before being treated with the BCA and challenged a second

139

time with P. expansion had significantly lower (p=0.0118) YM counts than P. expansum-

inoculated apples fumigated with PAA vapor before being treated with BCA and

challenged a second time with P. expansum. These results were unexpected, but

encouraging for small cider producers. While PAA is generally expensive, not readily

available (and when available, only in large quantities), and a potentially dangerous

product (PAA is a potent oxidizer and may damage skin when used full-strength), WV is

inexpensive, readily available in small quantities, and non-toxic, even if ingested.

Although WV was chosen for these trials after preliminary trials indicated a greater

efficacy associated with white vinegar than that associated with apple cider vinegar,

others (Sholberg and others 2000) have found that apple cider vinegar is effective against

P. expansum and other fungal pathogens, albeit slightly less so than white vinegar. It is

possible, for those so inclined, to produce cider vinegar from their own apple cider,

making their operation more vertically integrated and self-contained.

It was impossible to say whether the BCA improved any capacity of fumigations

to protect fruit from challenge with fungal pathogens, as no treatment group was designed

to assess protective capacity of fumigations alone. These experiments were designed to

determine whether fumigations improved the protective capacity of the BCA in storage.

We cannot say for certain, based on data collected in these experiments, if the BCA

improved protective capacity of the fumigations. Discerning whether the BCA treatment

enhanced the protective capacity of fumigations was not an objective, and treatment

classes were not designed to investigate this possibility. However, it seems logical to

conclude that, since the BCA was found to protect wounds against fungal infestation, and

since there is no reason to believe that fumigations provide any residual protection

140

against fungal infestation (Sholberg and others (2000) have found this to be true),

therefore, the BCA treatment could only augment the (lack of) protection provided by the

fumigations.

141

CONCLUSIONS

Treating apples with mild heat or fumigating them with vaporized PAA or WV

improved the ability of the Pseudomonas syringae BCA to control the growth of fungi in

wounds, during a short storage period of 2-3 weeks. White vinegar was overwhelmingly

most impressive in its ability to reduce populations of both extant fungi and inoculated

Penicillium expansion. The fact that it is possible to utilize a steam humidifier built for

home use to fumigate crops with vinegar is impressive. The CoolBot™, a small unit that

turns a window air conditioner into a refrigeration unit, is an example of how a similar

"off-label" use of a commonly available home product can offer numerous advantages to

small producers and can potentially reduce both overhead and production costs.

While there is currently no method of adequately removing patulin after it has

been formed, it is certainly possible to dramatically reduce patulin levels in cider by

taking measures to prevent production of patulin. In general, measures that improve

storage quality of apples (and cider) will tend to reduce patulin in cider. These measures

span both the apple growing season and the process of cider production, and include

adhering to cGAP and cGMP, taking steps to reduce disease in the field and diseases

caused by fungal pathogens during storage, and properly handling apples and cider after

processing. Fumigating apples with white vinegar and treating apples with a bacterial

biocontrol agent may be considered valid aspects of a plan to reduce patulin in cider,

when these measures are part of an integrated plan that includes the above mentioned

practices.

142

Integrated fungal pathogen control provides the best means for growers and

producers to adequately and efficiently combat fungal infestation and patulin

accumulation, with a modicum of resources. Effective integrated control involves

selecting disease resistant cultivars (currently no cultivars are resistant to P. expansum),

administering correct plant nutrition, irrigating crops appropriately (including avoiding

overhead irrigation), controlling fruit pests pre-harvest by applying appropriate

treatments throughout the season, harvesting fruit at the proper maturity for storage,

controlling fruit pests post-harvest by applying appropriate treatments to disinfect and

control fungal pathogens, maintaining good sanitation in packing lines and keeping dump

and flume water free of contamination, and storing produce under conditions that do not

favor pathogen growth (Sholberg and Conway 2001). An example of a process flow

diagram that integrates patulin reduction into the cider production process is included

here (Figure 10).

In light of the increasingly globalized agricultural market, an additional concern

raised by Moake and others (2005), involves problems arising when domestically

produced juices are blended with imported juices. This practice could be problematic for

a number of reasons. Food safety concerns may be elevated by the increasing

consumption of low-acid Chinese juices and juice concentrates, which may not

completely inhibit microbial growth or toxin formation. If producers make use of juice

from countries without regulations in place that mandate the same pre-production and

processing techniques as those in wide use throughout this country, then all control

measures taken have been effectively rendered moot.

143

Figure 10. Apple cider process flow diagram, showing where vinegar fumigation may be incoporated.

\ \ CCP Receiving

\ \

Pasteurization CCP

Wash n Wash Refrigerated Post-

Pasteurization Holding Tank

_ Refrigerated Post-Pasteurization Holding Tank

Inspection & Sorting/ Grading

Refrigerated Post-Pasteurization Holding Tank

Inspection & Sorting/ Grading

Refrigerated Post-Pasteurization Holding Tank

CCP CCP

Inspection & Sorting/ Grading

Refrigerated Post-Pasteurization Holding Tank

CCP CCP

Inspection & Sorting/ Grading

Refrigerated Post-Pasteurization Holding Tank

Inspection & Sorting/ Grading

Refrigerated Post-Pasteurization Holding Tank

\ X.

J Cold Storage ^ ^ CCP J Cold Storage ^ ^ r ^ Process Points for potential Vinegar

Fumigation

Filter/Screen CCP r ^ Process Points for potential Vinegar

Fumigation

Filter/Screen CCP

Brush/Rinse

r ^ Process Points for potential Vinegar

Fumigation ^

^ _ r - " " "

^

Mill/Grind Apples Aseptic Filling & Capping

Aseptic Filling & Capping

Press Pulp a Labelling CCP

Filter/Screen :s Cooler Storage

Refrigerated Pre-Pasteurization Holding Tank

Cooler Storage Refrigerated Pre-

Pasteurization Holding Tank

• CCP

Refrigerated Pre-Pasteurization Holding Tank

•

Refrigerated Pre-Pasteurization Holding Tank Delivery/Dispatch

^ . /

144

FUTURE RESEARCH

In these trials, we were looking for the greatest effect, and therefore trialed levels

of fumigant that predictably injured fruit. However, even in trial three, which used half

the level of fumigant, significant reductions of YM populations were still observed. We

did not determine a minimum inhibitory concentration (MIC), though this would be

interesting future work.

Vapor-phase AA has been shown to reduce levels of human pathogens on mung

beans (Delaquis and others 1999), and apples (Sapers and others 2003), although the

concentration of AA vapor used damaged apple fruit. Interesting future work might look

at using fumigations with WV to reduce populations of Cryptosporidium parvum, the

most resistant pathogen that is reasonably likely to be found in apple cider, as well as

finding fumigant concentrations that reduce populations of E. coli and other human

pathogens without damaging fruit.

Currently, FDA allows surface disinfection to constitute a portion of the 5 logio

reduction scheme for citrus fruit only. However, it would be interesting to determine

whether surface disinfection could safely contribute to a 5 logio total reduction of

potential pathogens in apple cider. It seems possible (but not likely) that pathogens could

persist within the core, but nonetheless, it would be interesting work to perform a risk-

benefit analysis concerning the use of surface disinfection in apple cider production.

Moake and others (2005) have alluded to the potential for enzyme degradation of

patulin and other mycotoxins; it would be interesting to determine if enzyme degradation

of patulin in cider is effective and possible from a cost perspective.

145

^m

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178

APPENDICES

APPENDIX A - PATULIN ANALYSIS

After sampling for microbial enumeration, five P. expansum-mocvXdiXQdi apples

from Trial three were held until blue mold was visible on substantial portions of the fruit.

At this point, the apples were considered to have been decayed by the fungi with which

they had been inoculated, a localized strain of P. expansum, and samples were analyzed

using reverse phase high performance liquid chromatography (HPLC) and atmospheric

pressure ionization tandem mass spectrometry (MS/MS) in series.

Five grams of decayed tissue was removed from each apple with a scalpel that

was sterilized by dipping in 95% EtOH and then passing through a flame in between each

incision. Patulin has been shown to be stable under mildly acidic conditions (Lovett and

Peeler 1973; Sapers and others 2005), and it was therefore determined that a weakly

acidic medium would best preserve patulin in the samples, and improve efficiency of

toxin recovery. The 5 g samples were placed in plastic conical centrifuge vials with 10

mL 0.1 % AA, and stored under refrigeration until HPLC extraction.

The first step of the HPLC analysis was to centrifuge the apple samples at high

speed for 10 minutes, pipette 5.0 mL of the supernatant into an eluting vial, and freeze

the remainder for any possible further analysis.

Next, a 60.0 mg Hydrophilic-Lipophilic-Balanced (HLB) column was attached to

a vacuum apparatus and conditioned, by running one column volume of methanol and

one column volume of 0.1 % acetic acid. An octydecylsilane stationary (reverse) phase

179

(C18-bonded silica) column was conditioned in the same manner, and the two were

connected in series, with the C18 column above the HLB column.

After conditioning the two columns, the 5.0 mL of the supernatant obtained by

centrifuging the P. expansum-decayed apple sample was loaded into the CI8 column, and

run through the two in series, by applying a vacuum. Once the entire sample had passed

through both the CI8 and HLB columns, the two columns were then washed in tandem

with 1.0 mL of 0.1 % acetic acid, and dried under vacuum for approximately 30 m.

Next, the two columns were disconnected from the vacuum apparatus and

separated, and a silica light sep-pak was attached to the bottom of the HLB column.

Through the HLB column and the silica light sep-pak, 1.5 mL ethyl acetate was eluted

into a small HPLC vial. This eluate was dried at 55 °C, in a sand bath, under a gentle

stream of N2.

The dried eluate was then re-dissolved with 1.0 mL of 0.1 % acetic acid, vortexed

for 30 s, and sonicated for 30 s, before being run into the HPLC-MS/MS setup. After

tuning the MS/MS for best ionizing conditions, an ion transition scan was run on a

patulin standard, to identify the fragments produced by negative ionization ("daughters")

(Figure Al). Then, using the results from the daughter scan, specific ion transitions

representing the fragmentation of patulin could be tracked by their retention time. Based

on identical HPLC retention times, identical ion transitions (fragments resulting from

negative ionization), and similar ratios of daughter ions to total ion chromatogram

(Figures A2 and A3), the HPLC-MS/MS results indicated that patulin was found at high

concentrations in apple tissue that had been decayed by the strain of P. expansum utilized

in these experiments.

180

Figure A.l. MS/MS spectrum showing daughter scan of patulin standard.

181

Figure A.2. Chromatogram of patulin standard (200 ppb), showing retention time and daughter ions.

Total Ion Chromatogram (patulin)

153 m/v - 124 m/v transition

153 m/v - 108 m/v transition

153 m/v - 136 m/v transition

Figure A.3. Chromatogram of sample, showing retention time and daughter ions.

182

APPENDIX B - COUNT DATA

The following tables represent the untransformed YM counts for every apple in

all three trials. CFU counts that appear in grey shaded cells have been estimated as 300;

the counts were then calculated by multiplying 300 by the appropriate dilution factor.

One (shaded cells are estimates). Table B.l . Data Tom Tria Trtmnt Apple CFU

wound"1

A 1 1300

A 2 1163

A 3 3300

A 4 1488

A 5 499 B 1 725 B 2 2200

B 3 160 B 4 151 B 5 48 B 6 525 B 7 16 B 8 44 B 9 16 B 10 594 CI 1 645 CI 2 2288

CI 3 574 CI 4 6400

CI 5 421 CI 6 852 CI 7 126 CI 8 234 CI 9 1825

CI 10 694 C2 1 0 C2 2 0 C2 3 0 C2 4 0 C2 5 0 C2 6 0

C2 7 0 C2 8 0 C2 9 0 C2 10 0 D 1 750000 D 2 17625

D 3 92500 D 4 83750

D 5 34125

D 6 348750

D 7 45250

D 8 750000

D 9 24250

D 10 17625

E 1 46 E 2 249 E 3 838 E 4 500 E 5 243 E 6 334 E 7 92750

E 8 11750

E 9 34000

E 10 403 Fl 1 841 Fl 2 15 Fl 3 50 Fl 4 51 Fl 5 1233

Fl 6 683 Fl 7 8 Fl 8 206 Fl 9 2100

apples

Fl 10 229 F2 1 0 F2 2 0 F2 3 0 F2 4 0 F2 5 0 F2 6 0 F2 7 543125

F2 8 0 F2 9 0 F2 10 0 Gl 1 41 Gl 2 30 Gl 3 1663

Gl _j 4 438 Gl 5 463 Gl 6 34 Gl 7 688 Gl 8 838 Gl 9 250 Gl 10 2338

G2 1 0 G2 2 0 G2 3 0 G2 4 . 0 G2 5 0 G2 6 0 G2 7 0 G2 8 0 G2 9 0 G2 10 0

Treatment A: Untreated control apples Treatment B: Apples that were treated with the biocontrol agent Treatment CI: Apples that were fumigated with peracetic acid solution Treatment C2: Apples that were fumigated with white vinegar Treatment D: P. exparawm-inoculated apples Treatment E: Apples treated with biocontrol agent prior to challenge with P. expansum

183

Treatment Fl: P. expansum-inoculated apples that were then fumigated with peracetic acid solution Treatment F2: P. expansum-inoculated apples that were then fumigated with white vinegar Treatment Gl: P. expansum-inoculated apples that were then fumigated with peracetic acid solution, and then treated with biocontrol agent Treatment G2: P. expansum-inoculated apples that were then fumigated with white vinegar, and then treated with biocontrol agent

184

Table B.2 . Data! Tom Tria Trtmnt Apple CFU

wound"1

A 1 204

A 2 314

A 3 328 A 4 609

A 5 393

A 6 95

A 7 106

A 8 276 A 9 216

A 10 86

B 1 4

B 2 11

B 3 4

B 4 314 B 5 155 B 6 60 B 7 30 B 8 1

B 9 1388

B 10 75000

C 1 0 C 2 0

C 3 0 C 4 0

C 5 0

C 6 153 C 7 0

C 8 0

C 9 0 C 10 3

D 1 156 D 2 0

D 3 19

D 4 126

D 5 103

D 6 1

D 7 1

D 8 461

D 9 70

D 10 1

E 1 261

E 2 771

E 3 5

E 4 156

E 5 61250

E 6 750000

E 7 750000

E 8 750000

E 9 750000

E 10 750000

F 1 2288

F 2 2625

F 3 13

F 4 1650

F 5 181

F 6 44

F 7 250

F 8 750000

F 9 500000

F 10 750000

G 1 0

G 2 3 G 3 0

G 4 0

G 5 0

G 6 0

apples

G 7 0 G 8 0 G 9 5 G 10 0 H 1 294 H 2 56 H 3 613 H 4 43 H 5 103 H 6 238 H 7 24 H 8 0 H 9 750000

H 10 750000

1 0 2 3 3 0 4 520 5 0 6 0 7 0 8 0

9 0 10 0

J 1 0 J 2 0 J 3 0 J 4 0 J 5 0 J 6 0 J 7 0

J 8 0 J 9 7500

J 10 7500

Treatment A: Untreated contro Treatment B: Apples that were treated with the biocontrol agent Treatment C: Apples that were fumigated with white vinegar Treatment D: Apples held at T < 32 °C overnight ("heat treatment") Treatment E: P. expansum-'m.ocx\\a\Q& apples Treatment F: Apples treated with biocontrol agent prior to challenge with P. expansum Treatment G: P. expaw^ww-inoculated apples that were then fumigated with white vinegar Treatment H: P. expansum-inocv\aXo,& apples that were then held at T < 32 °C overnight ("heat treatment") Treatment I: P. expattswwz-inoculated apples that were then fumigated with white vinegar, and then treated with biocontrol agent Treatment J: P. expa«s«m-inoculated apples that were then held at T < 32 °C overnight ("heat treatment"), and then treated with biocontrol agent

185

Table B.3 . Datal Tom Tria Trtmnt Apple CFU

wound"1

A 1 2175

A 2 2250

A 3 575

A 4 1950

A 5 23 A 6 2850 A 7 1025 B 1 587 B 2 180 B 3 149

B 4 204

B 5 93 B 6 168

B 7 49

C 1 0

C 2 0

C 3 1

C 4 0 C 5 0

C 6 0 C 7 0 D 1 1

Three (shaded cells are estimates). D 2 0

D 3 243

D 4 30

D 5 0

D 6 143

D 7 0

E 1 1325

E 2 330

E 3 45

E 4 43875

E 5 371250

E 6 273750

E 7 441250

F 1 285

F 2 170

F 3 384

F 4 134

F 5 9 F 6 243

F 7 173

G 1 0

G 2 0

G 3 0

G 4 0

G 5 0 G 6 0 G 7 0 H 1 27000 H 2 0 H 3 0 H 4 0 H 5 0 H 6 0 H 7 125000

1 0 2 0

3 0 4 0 5 0

6 0 7 7500

J 1 19 J 2 31 J 3 838 J 4 0 J 5 0 J 6 131250 J 7 125000

Treatment A: Untreated control apples Treatment B: Apples that were treated with the biocontrol agent Treatment C: Apples that were fumigated with white vinegar Treatment D: Apples that were fumigated with peracetic acid solution Treatment E: P. expansum-mocvAd&Qd. apples Treatment F: Apples treated with biocontrol agent prior to challenge with P. expansum Treatment G: P. expansum-'mocxxldLted apples that were then fumigated with white vinegar Treatment H: P. expansum-mocxAdXtd apples that were then fumigated with peracetic acid solution Treatment I: P. expansum-'moc\\\atQ& apples that were then fumigated with white vinegar, then treated with biocontrol agent, and then challenged again with P. expansum Treatment J: P. exp<msw/«-inoculated apples that were then fumigated with peracetic acid solution, then treated with biocontrol agent, and then challenged again with P. expansum

186

APPENDIX C - EFFECTS OF VARIOUS TRANSFORMATIONS ON DATA

The following tables summarize the data and the effects on the data of various

transformations.

Table C.l. Effects of transformations on data from Trial One.

Group Raw YM Count (CFU

per wound)

Logio -Transformed

Count

Square Root -Transformed Count

R a n k -Transformed Count

Mean Count

Rank Significance

Level Mean Count

Rank Mean Count

Rank Significance

Level Mean Count

Rank Significance Level

A 1.6 x 103 4 B 3.11 3 37.70 4 B 70.2 2 B

B 4.5 x 102 8 B 2.18 8 16.76 8 B 48.7 7 D

CI 1.4 x 103 5 B 2.89 5 32.24 5 B 62.9 3 BC

C2 0.0 x 10°

10 B 10 0.00 10 B 1.0 10 E

D 2.2 x 105 1 A 4.92 2 373.48 1 A 88.6 1 A

E 1.4 x 104 3 B 3.08 4 72.51 3 B 62.0 4 BCD

Fl 5.4 x 102 7 B 2.23 6 18.64 7 B 51.0 6 CD

F2 5.4 x 104 2 B 5.73 1 73.70 2 B 10.2 8 E

Gl 6.8 x 102 6 B 2.47 7 22.02 6 B 54.6 5 BCD

G2 0.0 x 10°

10 B 10 0.00 10 B 1.0 10 E

Treatments with the same Significance Level are not significantly different. (Significance levels not reported for the Logio transformation as this transformation results in undefined values for any YM counts of zero, preventing adequate statistical analysis.) Treatment A: Untreated control apples Treatment B: Apples that were treated with the biocontrol agent Treatment CI: Apples that were fumigated with peracetic acid solution Treatment C2: Apples that were fumigated with white vinegar Treatment D: P. expansum-'moculaXedi apples Treatment E: Apples treated with biocontrol agent prior to challenge with P. expansum Treatment Fl: P. expansum-'\nocx\\&\z& apples that were then fumigated with peracetic acid solution Treatment F2: P. ex/raraww-inoculated apples that were then fumigated with white vinegar Treatment Gl: P. expansum-mocu\sd.e.di apples that were then fumigated with peracetic acid solution, and then treated with biocontrol agent Treatment G2: P. exparaw/w-inoculated apples that were then fumigated with white vinegar, and then treated with biocontrol agent

187

Table C.2. Effects of transformations on data from Trial Two.

Group Raw YM Count (CFU

per wound)

Logio -Transformed YM Counts

Square Root -Transformed YM

Count

R a n k -Transformed YM

Count Mean Count

Rank Significance

Level Mean Count

Rank Mean Count

Rank Significance Level

Mean Count Rank Significance

Level

A 2.6 x 102 6 c 2.34 5 15.5 6 c 68.7 3 ABC

B 7.7 x 103 4 c 1.83 6 36.3 4 c 57.9 5 CD

C 1.6 x 101 9 c 1.29 9 1.4 9 c 10.9 9 E

D 9.4 x 10"

7 c 1.38 8 7.1 7 c 47.0 6 D

E 3.8 x 105 1 A 4.24 1 463.6 1 A 81.0 1 A

F 2.0 x 105 2 B 3.48 3 261.8 2 B 76.5 2 AB

G 7.5 x 10"' 10 C 0.55 10 0.4 10 C 9.1 10 E

H 1.5 x 105 3 BC 2.91 4 181.8 3 BC 61.9 4 BCD

I 5.2 x 101 8 C 1.56 7 2.4 8 C 12.6 8 E

J 1.5x 103 5 C 3.88 2 17.3 5 C 18.2 7 E

Treatments with the same Significance Level are not significantly different. (Significance levels not reported for the Logio transformation as this transformation results in undefined values for any YM counts of zero, preventing adequate statistical analysis.) Treatment A: Untreated control apples Treatment B: Apples that were treated with the biocontrol agent Treatment C: Apples that were fumigated with white vinegar Treatment D: Apples held at T < 32 °C overnight ("heat treatment") Treatment E: P. exparawm-inoculated apples Treatment F: Apples treated with biocontrol agent prior to challenge with P. expansum Treatment G: P. expansum-mocxAated. apples that were then fumigated with white vinegar Treatment H: P. expansum-inocvXated apples that were then held at T < 32 °C overnight ("heat treatment") Treatment I: P. ex/?araum-inoculated apples that were then fumigated with white vinegar, and then treated with biocontrol agent Treatment J: P. expansum-'moc\\\atc& apples that were then held at T < 32 °C overnight ("heat treatment"), and then treated with biocontrol agent

188

Table C.3. Effects of transformations on data from Trial 3. Group Raw YM Count (CFU

per wound) LoglO -

Transformed YM Count

Square Root -Transformed YM

Count

R a n k -Transformed YM

Count Mean Count

Rank Significance Level

Mean Count

Rank Mean Count

Rank Significance Level

Mean Count

Rank Significance Level

A 1.6 x 103

4 B 2.94 5 36.1 4 B 54.4 2 AB

B 2.0 x 102

6 B 2.20 6 13.4 5 B 44.1 4 B

C 1.8x 10"'

9 B 0.10 9 0.2 9 B 5.1 9 D

D 5.9 x 101

8 B 1.53 8 4.9 8 B 22.4 6 CD

E 1.6 x 105

1 A 4.08 2 295.4 1 A 59.4 1 A

F 2.0 x 102

7 B 2.14 7 13.2 6 B 44.4 3 B

G 0.0 x 10°

10 B 10 0.0 10 B 1.0 10 D

H 2.2 x 104

*» j B 4.76 1 74.0 3 B 19.0 7 CD

I 1.1 X 103

5 B 3.88 3 12.4 7 B 9.7 8 D

J 3.7 x 104

2 B 3.18 4 107.8 2 B 36.9 5 BC

Treatm ents w ith th< i same Sigi lificanc 3 Leve are no t sign ificantly differenl t. (Significance levels not reported for the Logio transformation as this transformation results in undefined values for any YM counts of zero, preventing adequate statistical analysis.) Treatment A: Untreated control apples Treatment B: Apples that were treated with the biocontrol agent Treatment C: Apples that were fumigated with white vinegar Treatment D: Apples that were fumigated with peracetic acid solution Treatment E: P. expansum-inoculated apples Treatment F: Apples treated with biocontrol agent prior to challenge with P. expamum Treatment G: P. expansum-inoculated apples that were then fumigated with white vinegar Treatment H: P. expansum-inoculated apples that were then fumigated with peracetic acid solution Treatment I: P. expansum-inoculated apples that were then fumigated with white vinegar, then treated with biocontrol agent, and then challenged again with P. expamum Treatment J: P. expansum-inoculated apples that were then fumigated with peracetic acid solution, then treated with biocontrol agent, and then challenged again with P. expamum

189

BIOGRAGPHY OF THE AUTHOR

Lucius Caldwell was born in Cohasset, Massachusetts. He has a Bachelor of

Science degree from McGill University, Montreal, Canada, where he studied Biology and

Philosophy. He then came to Maine to pursue a Master of Science degree in Food

Science and Human Nutrition at The University of Maine, beginning in September 2007.

Upon completion of the Master of Science degree at The University of Maine, he will

start a Ph.D. program in Biology at the University of Idaho, Moscow.

Lucius Caldwell is a candidate for the Master of Science degree in Food Science

and Human Nutrition from The University of Maine in August 2009.

190