Development of a heat treatment to enhance the antimicrobial properties of wood based mulches and

animal bedding materials

By

Mina Kalantarzadeh

UNIVERSITY OF

SURREY

SUBMITTED FOR THE DEGREEOF

DOCTOR OF PHILOSOPHY

N A T U R A L PR O D U C T S R ESE A R C H G R O U P

D E PA R T M E N T O F C H E M IST R Y

FA C U L T Y O F E N G IN E E R IN G A N D PH Y SIC A L SC IE N C E S

U N IV E R SIT Y O F SU R E R R Y

D ecem ber 2013

© M. K

ProQuest Number: 27598828

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Acknowledgements

I would like to express my sincere gratitude to my supervisor. Professor Dulcie Mulholland,

for their support, time and guidance through my PhD. She provided me the opportunity o f

working in Natural Product Group which I will forever appreciate. I would not be able to

continue with my PhD project if she would not stand by me all along the changes and

difficulties during my study at University o f Surrey. I thank you Professor M ulholland for

your generous and loving heart.

I also would like to particularly thank Dr Joan Webber, the project manager for

Phythophthora control programme in Forest Research for their continuous support during the

work on P. ramorum. I appreciate the time and effort she put in supervising me with drafting

my thesis. I thank you Dr W ebber for your understanding and consideration. I should also

thank my former and co-supervisors Dr Frans de Leij and Professor La Ragione for the time

that they lent to me.

I also wish to thank Dr Moses Langat and Dr Dan Driscoll at Chemistry Department,

University o f Surrey, for their help and supervision with NM R spectroscopy and GC-MS

analyses o f the wood materials that are the core to this study. Appreciation also goes to the

members o f Pathology Department at Forest Research, who were all kind supportive and

welcoming particularly M r Andy Jeeves, for their laboratory technical support and M r Tony

Reeves for their kind and priceless support with the field trials in Cornwall. I would like to

thank the head o f Land Regeneration and Urban Greenspace at Forest Research, M r Tony

Hutchings and Ms Vicki Lawrence for their help and support.

The journey through this PhD life has enabled me to meet with people from all parts o f the

world. Many o f whom I have had a chance to make friends with. I would like to thank the

following people for their support and encouragement during the past three years, W atcharee

W aratchareeyakul, Katerina Ridge and Dorota Nawrot. I always will remember you.

I would like to thank my family members, specially my daughter and my sisters for their

love, patience and consideration during this journey. I would like to thank Robert and his

family for their support.

Finally, I would like to acknowledge Forest Research Centre (Alice Holt Lodge) for funding

my research.

IV

Table of content

Declaration............................................................................................................................... ii

Dedication .........................................................................................................................iii

Acknowledgements........................... iv

Table of content................................................ v

List of figures.................................................................................................................. xi

List of tables............................................ xiii

List of abbreviations..............................................................................................................xv

Abstract...................................................................................................................................xvi

CHAPTER 1 : INTRODUCTION...........................................................................................1

1.1 Heat-treated woods and their applications......................................................................1

1.1.1 Characteristics of heated wood m aterials................................................................... 2

1.1.2 Antimicrobial chemicals................................................................................................2

1.1.3 Chemical characteristic of wood before and after heating........................................ 3

1.1.3.1 Wood extractives and their chemistry......................................................................3

1.1.3.2 Aliphatic compounds..................................................................................................4

1.1.3.3 Terpenes and terpenoids........................ 4

1.1.3.4 Phenolic extractives.................................................................................................... 5

1.1.3.5 Wood structural components...................... 6

1.1.4 Changes in thermally modified w ood................................................................... 8

1.1.4.1 Hydrolysis of wood structural components as a result o f heating...................... 8

1.1.4.2 Colour change............................................................................... 9

1.1.4.3 Formation of acid........................... 9

1.1.5 Novel applications for heat treated w ood................................................................. 10

1.2 Background to poultry production and its economic significance............................10

1.2 Heath risks associated with poultry .........................................................................11

1.2.1 Salmonellosis and public health..................................................................................11

1.2.1.1 Broiler production and associated risk factors of Salmonellosis .......... 12

(1) Broiler production .......................................................................................................... 12

(2) Major sources of Salmonella in broiler houses............................................................13

1.2.1.2 Significance of litter moisture on Salmonella survival......................................... 14

1.2.1.3 Control of Salmonella in the gastrointestinal tract of growing broilers............ 15

(1) Competitive exclusion......................................................................................................15

(2) Probiotics ......... 16

(3) V accines.............................................................................................................................16

(4) Biosecurity measures................................................................. 16

1.2.1.4 Litter control m easures.......................................................................................... 17

Moisture control .......................................................... 17

Litter pH control.......................................................................................... 17

Bedding m aterials............................................................................. .:...................................18

1.2.2 Ammonia emissions..................... 18

1.2.2.1 Animal welfare and human health....................................................... 18

1.2.2.2 Environmental impacts of ammonia emission from broiler houses...................19

1.2.2.3 Production of ammonia in broiler un its............... 20

1.2.2.4 Ammonia production control and litter management..........................................21

(1) Moisture control............................................................................................................... 21

(2) Litter amendments............................................................................................................22

(3) Characteristics of wood materials as animal bedding m aterials ......................... 23

1.2.2.5 Limitation in using bedding materials and alternative approaches....................24

1.3 Background to the impact and biology of Phytophthora ramorum.................... ..25

1.3.1 The causal agent of Sudden Oak Death: Phytophthora ramorum ........................26

1.3.2 Symptoms caused by Phytophthora ramorum................................ 27

1.3.3 Risk factors of Phytophthora ramorum....................................................................28

1.3.3.1 Distribution................................................................................................................ 28

1.3.3.2 Host factors.............................. 29

(1) Range of Plant hosts........................................................................................................ 29

(2) Role of foliar hosts on disease cycle and development.............................................. 30

1.3.3.3 Environmental factors............................................................................................ 31

1.3.4 D ispersal........................................................................................................................32

1.3.5 Survival.................................................................... 32

1.3.6 Disease current management......................................................................................33

1.3.6.1 Exclusion..................................................................... 33

1.3.6.2 Best management practice.......................................................................................34

1.3.6.3 Eradication.......................................................................................................... 34

VI

1.3.6.4 Heat treatment.................................................... 35

1.3.6.5 Chemical treatments ................... 35

1.3.6.6 Natural products.........................................................................................................36

1.4 Summary and research objectives...............................................................................37

CHAPTER 2: MATERIALS & METHODS..................................................... 38

2.1 Bacterial isolates and media ............................................... 38

2.1.1 Solid agar m edia.............................. 38

2.1.2 Liquid growth media.................................. ...38

2.1.3 Bacterial enumeration (cfu ml’^)................................................................................ 38

2.2 Thermal treatment technique.......................................................................................38

2.2.1 Wood Materials...................................................... 38

2.2.2 Wood treatment temperature.......................................................................................39

2.2.3 Wood treatment time.................................................................................................... 39

2.2.4 Wood species................ 39

2.3 CO2 production............................ 39

2.4 Production of ammonium ions.......................................................................................40

2.5 pH ....................................................................................................................................... 40

2.6 Chemical analysis techniques........................................................................................40

2.6.1.1 Wood extraction.........................................................................................................40

2.6.1.2 Water extraction.........................................................................................................41

2.6.3 Methanol extraction...................................................................................................41

2.7 GC-MS analysis o f heated and non-heated wood shavings ............................. 41

2.8 Isolation of active compounds derived from the methanol crude extract............... 41

2.8.1 Thin layer chromatography .............................................................................. 41

2.8.2 Column chromatography............................................................................................ 41

2.9 In vitro assays.................................................................................................................. 42

2.9.1 Direct contact assay .................................................... 42

2.9.2 Synergistic effect assay........................... ...42

2.9.3 pH activity assay...........................................................................................................43

2.9.4 Bioautographic T L C ............................................................................................. 44

2.10 Determination of the effect of heating of rhododendron and larch wood shaving on P. ramorum............................................... 44

Vll

2.10.1 Wood species of tested materials.............................................................................44

2.10.2 Preparation of crude extracts................................................................... 44

2.10.3 Test isolates................................................................................................................. 44

2.10.3.1 Inoculum preparation............... 45

2.10.4 vitro assays.............................................................................................................46

CHAPTER 3: EVALUATION OF HEAT-TREATED WOOD AS LITTER .............47

3.1 Introduction........................................................ 47

3.2 Experimental.....................................................................................................................49

3.2.1 Wood inoculation..........................................................................................................49

3.2.2 Litter model preparation............................................................................................. 49

3.3 Results........................................................... 50

3.3.1 Effect of heating regime on the anti-microbial properties of pine bark............... 50

3.3.2. Effect o f heated pine shavings on microbial breakdown in modelled litte r 51

3.3.3 Effect of heated pine wood on CO2 production from wood shavings based litter (as indicator of microbial activity)............................................................................................ 52

3.3.4 Effect of heating duration at 120°C on CO2 production, pH and ammonia concentration ................. 53

3.3.5 Spectrum o f enhanced antimicrobial activity of heated pine shavings................ 56

3.3.6 Evaluation of the antimicrobial activity o f different wood types after heating ..57

3.3.7 Effect o f time of exposure to heat treatment of 120°C on pH of pine wood shavings 57

3.4 Discussion................................................................................................................... ...58

CHAPTER 4; CHEMICAL ANALYSIS AND EVALUATION OF ENHANCED ANTIMICROBIAL ACTIVITY OF HEAT-TREATED WOOD.................................................................... 62

4.1 Introduction.......................................................................................................................62

4.2 Experimental.....................................................................................................................64

4.2.1 Extract assays......................... 64

4.2.2. Chemical analysis........................................................................................................65

4.3 Results................................................................................................................................65

4.3 .1 Extract assays of the methanol crude extract........................ 65

4.3.2 GCMS analysis of crude extracts of p in e .................................................................66

4.3.3 Active fractions from column chromatography ................... 67

4.3.4 Yield of different molecules o f interest from heated Scots pine wood shavings 68

4.3.5 Synergistic effect o f dehydroabietic acid on the antimicrobial activity of vanillin69

v i i i

4.3.6 Effect of low pH on antimicrobial activity o f vanillin using citric acid ..............70

4.3.7 Antimicrobial activity of commercial vanillin and D H A A ...................................70

4.3.8 Effect of pH and storage temperature on antimicrobial activity o f commercial vanillin/DH AA..................................... 71

4.3.9 Spectrum of antimicrobial activity o f the methanol crude extract and isolated fractions of heated wood of pine............................................................... 72

4.4 Discussion.............................................................................. 72

CHAPTER 5: EFFECT OF HEATED WOOD ON P. RAMORUM ............................. 76

5.1 Reasons for concern.........................................................................................................76

5.2 Alternative approaches,to control............... 76

5.2 Experimental.....................................................................................................................79

5.2.1 Micro-cosm trails..........................................................................................................79

5.2.1.1 Effect of larch, pine and rhododendron heated woodchips on the survival of Phytophthora ramorum zoospores.......................................................................................79

5.2.1.2 Effect o f incorporating heated woodchips made from larch into soil infected with P. ramorum................... ...79

5.2.1.3 Effect of incorporated heated woodchips made from larch on the survival o f P. ramorum chlamydospores.......................... 80

5.2.2 In vitro assays........................................... 81

5.2.2.1 Assessment o f Minimum Inhibitory Concentration (MIC) of crude extracts against P. ramorum zoospores................................................................................................................ 81

5.2.2.2 Effect of crude extracts derived from heated and air-dried wood on chlamydospore germination of P. ramorum................................................................................................... 81

5.2.2.3 MIC of chemicals in crude extracts derived from heated pine wood against hyphae, zoospores and chlamydospores o f P. ramorum................................................. 82

5.2.3 Field studies............................................................... 82

5.2.3.1 Largin Wood field evaluation........................ 82

5.2.3.2 Druid’s Hill field evaluation....................................................................................83

5.2.4 Chemical analyses.........................................................................................................86

5.2.4.1 Chemical analysis of heated larch, pine and rhododendron wood.....................86

5.2.4.2 Chemical analysis of discoloured bark of a Japanese larch................................86

5.3 Results............................................................................................................................. 87

5.3.1 Microcosm studies........................................................................................................87

5.3.1.1 Evaluation of woodchips derived from larch, pine and rhododendron............. 87

IX

5.3.1.2 Effect o f incorporating heated woodchips into soil infested with zoospores ..88

5.3.1.3 Effect of incorporated heated larch woodchips on the survival of chlamydospores 88

5.3.2 In vitro assays............................ ...............................................................................89

5.3.2.1 Assessment o f minimum inhibitory concentration (MIC) of crude extracts against zoospores..................................................................................................................................89

5.3.2.2 Effect of crude extracts derived from heated and air-dried wood on chlamydospore germination............................................................................................................................. 89

5.3.2.3 Minimum Inhibitory Concentration (MIC) of different chemicals found in crude extracts derived from heated pine w ood...................... ...................................... ...............90

5.3.3 Field studies..................................................................................................................91

5.3.3.1 Largin Wood evaluation..........................................................................................91

5.3.3.2 Druid’s Hill field evaluation................................................................................... 92

5.3.4 Chemical analyses........................................................................................................ 93

5.3.4.1 Chemical analysis of heated larch, pine and rhododendron wood used in the field 93

5.3.4.2 Chemical analysis of discoloured bark of a Japanese larch............................... 93

5.4 Discussion...................... ....96

CHAPTER 6: DISCUSSION..............................................................................................103

Future w ork....................... 110

CHAPTER 7: REFERENCES.............................. ............................................................ 112

Appendices................................................................................................................... 128

List of figures

Figure 1.1 Examples of monoterepenes and their derivatives in softwood ................. 4

Figure 1.2 Examples o f resin acids ...............................................................................................5

Figure 1.3 Examples of phenolic extractives........................................................... 6

Figure 1.4 Arrangements of fibrils, microfibrils and cellulose in cell w a lls ...............................6

Figure 1.5 The most important hemicelluloses in softwoods; xylan and glucomannan............7

Figure 1.6 Structure of softwood lignin............................................................................................ 7

Figure 1.7 Examples of chromophores in wood according to Sundqvist, 2004.......................... 9

Figure 1.8 A broiler growing house................................................................................................. 13

Figure 1.9 Illustration of processes related to ammonia emissions from litter based manure 21

Figure 1.10 Symptoms of sudden oak disease in woodland and forest...................................... 26

Figure 1.11 Sporulation of Phytophthora ramorum...................................................... 27

Figure 1.12 The symptoms of infection by Phytophthora ramorum ..........................................28

Figure 1.13 The disease cycle of P. ramorum ............................................................................... 31

Figure 2.1 The methanol extracts of heat-treated (left) and air-dried (right) wood shavings of pine......................................................................................................................................................... 45

Figure 3.1 Litter models made in 2.5 liter plastic containers...................... 50

Figure 3.2 Change of pH in modelled litter over time.............................. 52

Figure 3.3 Comparison o f pH between 8 different model litters over time (n=4).................... 55

Figure 3.4 Changing pH values of pine shavings after different times of heat treatment........58

Figure 4.1 A comparison between two GC-MS chromatograms of heated and air dried pine wood.......................................................................................................................................................67

Figure 4.2 Compounds isolated from Pinus sylvestris after heat treatment at 120°C..............69

Figure 5.1 Dead larch trees infected by Phytophthora ramorum ............................................... 76

Figure 5.2 Larch trees infected by Phytophthora ramorum, Crymant, near N eath ................. 77

Figure 5.3 Field site in Largin Wood....................................................................... 84

Figure 5.4 Example of trays with wood chips placed underneath a rhododendron bush at Druid’s Hill, Cornwall............... 85

Figure 5.5 Bark (phloem) of a Japanese larch tree showing different stages of discolouration. ; 86

Figure 5.6 Examples of an assay plate (96-well plate) after the chemically treated zoospores are plated on SMA (a), and (b) an enlarged picture of normal growth of P. ramorum in sterile water (control)..............................................'....................................................................... 90

Figure 5.7 Comparison of mass spectra of methanol crude extracts of (a) heated pine (b) larch and (c) rhododendron..........................................................................................................;..... 94

Figure 5.8 Comparison o f mass spectra of crude extract o f discoloured bark from Japanese larch........................................................................................................................................................ 95

XI

Figure 6.1 Interactions of secondary metabolites with biomembrane and membrane proteins. 108

XU

List of tables

Table 1.1 Examples of aliphatic extractives in xylem and bark.....................................................4

Table 3.1 Effect of different heating temperatures on the survival o f Salmonella Enteritidis in ground pine bark ......................................................................................................................50

Table 3.2 Effect of source materials on the survival of Salmonella Enteritidis in pine shavings before and after heat treatment..................................................... ;................................... 51

Table 3.3 Effect of different heating temperature on the survival of Salmonella Enteritidis o n , filter paper........................................................................................................................ 51

Table 3.4 Effect of heating at 150°C and 200°C on the pH of pine shavings made into litter. ........................................................................................................................ 52

Table 3.5 Effect of heating at 150°C and 200°C for one hour on the microbial activity expressed as CO2 production in a pine shavings base litter...........................................................53

Table 3.6 Effect of heating pine shavings at 120°C for increasing periods of time (1 to 72 hours) on antimicrobial activity expressed as CO2 production..................................................... 54

Table 3.7 Effect o f heating pine shavings at 120°C for increasing periods of time (1 to 72 hours) on antimicrobial activity of the treated shavings expressed as the pH .............. 54

Table 3.8 Effect o f heating pine shavings at 120°C for increasing periods of time (1 to 72 hours) on antimicrobial activity of the treated shavings expressed as ammonium ion concentration .................................................................................... 56

Table 3.9 Survivals of S. aureus, and E. coli and on heat treated and air-dried wood o f pine and air dried and heated filter paper. ..............................................................................................56

Table 3.10 Recovery o f Salmonella Enteritidis from inoculated heated and air-dried wood shavings derived from 8 different wood species............................................................................. 57

Table 4.1 Recovery of SE from inoculated filter paper treated with extracts from heated and air-dried pine shavings.........................................................................................................................65

Table 4.2 Recovery of Salmonella (log cfu g' ̂± SE) from inoculated, heated and air-dried pine shavings..................................................... 66

Table 4.3 Yield of different compounds isolated from heated wood o f P. sylvestris...............68

Table 4.4 A comparison of antimicrobial activity of vanillin and combinations of vanillin and dehydroabietic acid on the survival O f SE.............................................................. 70

Table 4.5 Effect of citric acid on the antimicrobial activity of vanillin against Salmonella Enteritidis...............................................................................................................................................71

Table 4.6 Antimicrobial activity of commercial vanillin alone and in combination with dehydroabietic acid on the survival of Salmonella Enteritidis......................................................71

Table 4.7 Effect of pH and temperature on antimicrobial activity of on vanillin/ DHAA solution...................................................................................................................................................72

Table 5.1 Survival of P. ramorum zoospores inoculated on air-dried and heated woodchips. ............................................................................................................................................. 87

Table 5.2 Effect of incorporation rate of heated larch woodchips into soil on the survival oftwo isolates of P. ramorum, 3 days and 8 weeks after inoculation (n=3).....................................87

X lll

Table 5.3 Effect of heated woodchips of larch on the germination o f leaf disks of rhododendron....................................... 88

Table 5.4 Minimum inhibitory concentration o f the methanol crude extract (mg ml'^) of air- dried and heated wood of pine, larch and rhododendron (n=3).............................. ....89

Table 5.5 Effect of methanol crude extracts of air-dried and heated wood o f pine, larch and rhododendron on germination o f pre-colonized leaf disks o f rhododendron with chlamydospores of two isolates of P. ramorum.................................................................. 90

Table 5.6 Minimum inhibitory concentration (mg mf^) of the compounds derived from heated wood of pine on mycelia, zoospores and chlamydospores (in colonized leaf disks) of two isolates o f P. ramorum................................................................................................................. 90

Table 5.7 Minimum inhibitory concentrations of acetovanillone, coniferaldehyde and vanillin, with and without 20 mM dehydroabietic acid (DHAA) tested against zoospores of P. ramorum (n=3).............................................................................. 91

Table 5.8 Minimum inhibitory concentration o f acetovanillone, coniferaldeyde and vanillin with or without either 20mM of abietic, dehydroabietic or pimaric acid tested against zoospores of P. ramorum isolate B RCO l........................................................................................ 91

Table 5.9 Effect of heat treatments on survivals of P. ramorum on woodchips of larch, rhododendron and pine .......................................................................................................... 92

XIV

List of abbreviations

N M R spectroscopy proton nuclear magnetic resonance spectroscopy

AA abietic acid

ASPs acid shock proteins

ATR acid tolerance response

Aw Water Activity

CDC Centre for Disease Control and Prevention

CE compétitive exclusion

cfli colony forming unit

cm centimetre

DCM dichloromethane

DEFRA/defra department for environment, food and rural affairs

DHAA dehydroabietic acid

EFSA European Food Safety Authority

g gram

ha hectare

IPPC Integrated Pollution Prevention and Control

L litre

m/z mass/charge ratio (mass spectrometry)

MIC minimum inhibitory concentration

ml millilitre

mmol millimolar

NIST NIST mass spectral library

NM R nuclear magnetic resonance spectroscopy

PA pimaric acid

R .0 reverse osm osis

RT-PCR real-time polymerase chain reaction

SMA standard method agar

TLC thin layer chromatography

U SDA United States Department o f Agriculture

XLD xylose lysine deoxycholate agar

pL microliter

XV

Abstract

Heat treatment is the most commonly practiced technology to add value to products derived

from wood materials. Traditionally, heat treatment enhances the resistance of wood materials

to weathering and decay fungi, but despite its wide use in sectors such as construction, wood

preservation and food packaging, there has been little attempt to apply heat treatment in other

areas to manage food-home and plant diseases. A preliminary study indicated that pre­

heating increased the inhibitory effect o f pine wood shavings against the important food-

home pathogen o f broilers, Salmonella Enteritidis (SE). Thus the first objective of this study

was to optimise a heating regime to evaluate the effect o f the enhanced inhibitory activity on

SE in direct contact and also reducing ammonia emission of a constructed litter using heated

wood. Heating to 120°C for 72 hours was the most effective treatment. In addition, heated

pine wood had antimicrobial activity against other known microorganisms in poultry bedding

including Staphylococcus aureus, Escherichia coli and Saccharomyces cerevisiae as well as

SE. Hardwood shavings of ash, beech, red oak also showed antimicrobial activity against SE

as did similarly treated softwood species such as pine and cypress. In vitro assays of

dominant chemicals derived from fractionation o f the extract demonstrated that bio-active

fractions containing vanillin and dehydroabietic acid exhibited antimicrobial activity, but

weak or no activity after purification. Overall, the in vitro assays suggested that a synergistic

interaction occurred between vanillin and dehydroabietic acid decreasing the minimum

inhibitor levels of vanillin. A second objective of the study was to investigate the potential of

heat-treated woodchips of Japanese larch and rhododendron to control Phytophthora

ramorum (PR) an invasive pathogen of larch in UK forests. Results demonstrated that heat-

treated pine, larch and rhododendron woodchips inhibited the recovery o f zoospores o f PR

compared with air-dried woodchips. This inhibition was maintained even if the larch

woodchips were diluted with soil. The in vitro assays revealed that their methanol cmde

extracts had an inhibitory effect on PR zoospores and also reduced germination of

chlamydospores compared with extracts from air-dried wood. Chemical analysis o f crude

extracts of all three showed some of the induced compounds were present in all the extracts

but differed in concentration. Coniferaldehyde was the most active compound against all

three propagules. The dominant resin acids, DHAA and abietic acid (AB) decreased the

minimum inhibitory level of the tested phenolic compounds against PR but had no effect

when tested alone. Results of a field trial using heat-treated and air-dried woodchips were

consistent with the bioassay results of crude extracts and indicated that heat treated materials

have potential to reduce the survival of PR under natural conditions. Finally, based on

XVI

overall results of bioassays in both parts of this study a mechanism of action for the

synergism between aromatic compounds such as vanillin and coniferaldehyde and resin acids

dehydroabietic and abietic acid was hypothesized.

XVll

CH APTER 1; INTRO DUCTIO N

1.1 Heat-treated woods and their applications

Wood and wood products are the most important source of renewable bio-mass and they have

a wide range of applications. The versatile nature of wood makes it possible for processing

industries to produce a vast number o f products with different applications. However, wood

materials are likely to become the habitat o f different microorganisms such as pathogenic

bacteria and moulds because of their porous structure. They can also be susceptible to decay

fungi. Wood materials are sanitised using different techniques such as washing, radiation

steam and dry heating. Dry heating techniques are widely used in industry as simple and

economic techniques which give heat-treated materials an advantage over raw material in

use. These heated wood materials have a wide range of different applications, in construction

and furniture, musical instruments, children playgrounds and food packaging.

In construction and furniture manufacture, heating is mainly used to improve wood

dimensional stability and its resistance to weathering conditions and decay fungi. Heating is

commonly used to improve hygiene of wood materials in contact with food (Beyer and

Gudbjomsdottir, 2002; Sjostrom, 1981). Wood packaging materials are required to be heat-

treated to prevent the spread of harmful microorganisms.

These improvements have been shown to be due to physical and chemical changes taking

place during heating and depend on two critical factors, temperature and time o f exposure

(Esteves et a l, 2008; Sundqvist, 2004). Lower temperatures improve hygiene conditions of

wood simply by making the wood a less favourable substrate for colonization by microbes.

According to the FAO, a core heat treatment of 56°C for a minimum of 30 minutes is

essential to pasteurize wood materials used in food packaging (FAO, 2002). Meat handling

pallets and vegetable boxes are heat-treated to clean the raw materials against mould before

use (Gudbjomsdottir et ah, 2000). Microwave techniques can also be used to sanitise

wooden pallets and egg trays and boxes (Gudbjomsdottir et a l, 2000). Examples include egg

trays heated at 90°C to pasteurise them and prevent transmission of poultry pathogens, such

as Salmonella, between sites (Gudbjomsdottir et a l, 2000). High temperatures of 100-115°C

are effective at reducing food pathogenic agents. Wooden kitchen utensils and cutting boards

are usually heat treated at the end of manufacturing (Beyer and Gudbjomsdottir, 2002). High

temperature treatments are also applied when a high degree of resistance toward target

microorganisms is required in products, such as outdoor fumiture and construction-aimed

materials. Temperatures above 200°C are usually known to be effective with these kinds of

applications (Esteves et a l, 2008; Kamdem et a l, 2002). When wood materials are heated at

very high temperatures they can be chemically modified and exhibit different characteristics

depending on the type of process applied (Esteves et a l, 2008; Esteves et a l, 2011).

1.1.1 Characteristics of heated wood materials

Heat treatment is one o f the wood industry’s main processes used to change the colour,

improve durability to biodégradation and weathering and to improve dimensional stability of

wood (Sundqvist, 2004). Among these, durability o f thermally modified wood against fungi

is of a great interest. Heating of wood seems to be an effective method to improve its

resistance against fungal attacks. It has been found that heating at 200°C, leads to wood

resistance against blue-stain, white-rot and brown-rot fungi (Kamdem et al, 2002; Savluchinske-

Feio et al, 2007). Some heat treatment techniques are also shown to have the potential of

replacing wood preservatives with high environmental impacts (Savluchinske-Feio et a l,

2007). Thermally modified woods show improved antimicrobial activities compared to

untreated sources. Previous researches indicate that the bioactivity is partly due to new

chemicals induced in wood materials after heating (Esteves et a l, 2008; Kamdem et a l,

2002; Sundqvist, 2004). In some cases, identified compounds are known for their potential in

a variety o f other antimicrobial applications and are produced from wood in large quantities

by heating techniques (MacKay et a l, 2009a; Murzin et a l, 2007).

1.1.2 Antimicrobial chemicals

There are literally hundreds o f chemical products derived from wood species which are used

in medicines, as food additives, in cosmetics, or act as preservatives, fungicides and

insecticides (Voda et a l, 2003; Wink, 2006). Chemical products o f wood are divided into

two general groups, wood extractives and chemicals derived from wood processing

techniques. Wood extractives are secondary metabolites which are extracted from raw

materials. Many of these show antimicrobial activity and are used widely as medicines, food

additives, spices, etc. Valuable chemicals derived from wood processing are also used as

food preservatives, many others are manufactured and are used as antimicrobial agents or

intermediate chemicals in the production of pharmaceuticals.

Wood processing chemicals are induced when wood undergoes different chemical reactions

upon different processing. Many o f these processes have been practiced for years and many

others have received substantial research and development efforts to introduce new products

or understand how to best obtain them. Depending on the type of heating process, wood

materials with different eharacteristics and chemicals are produced. These include high

energy processes such as pyrolysis and gasification that consume a lot o f energy in a range of

400°C or even higher. They are used to produce fuels or fuel intermediates such as Syngas

that is used to produce methanol, ammonia and bio-fuel. Some by-products of high energy

processing exhibit antimicrobial properties and are widely manufactured; such an example is

wood vinegar (MacKay et a l, 2009a; Williams, 2012). Wood vinegar is used in cosmetic

products such as shampoos and lotions and acts against bacteria and fungi that cause skin

damage (Williams, 2012).

Low energy processes like fermentation, extraction and torrefacation are applied to produce

different chemicals such as solvents and resins in large quantities. Some others are used to

produce bioactive chemicals with application in food flavourings, preservatives, pharmaceuticals

and their intermediate chemicals. Acetic acid is produced by either fermentation or dry

heating (different wood materials) and has application in the preparation o f pharmaceuticals

such as aspirin and some fungicides (MacKay et a l, 2009a). Phenolic aldehydes such as

syringaldéhyde and vanillin are pharmaceutical precursors (MacKay et a l, 2009a).

Syringaldéhyde is also used as hair and fibre dye. Vanillin is used as a preservative and

flavouring agent in food products such as cakes and ice creams. Others are used as smoke

flavours and meat preservatives, such as guaiacol and syringol. These chemicals are

produced as a result o f chemical modification in wood structural components (MacKay et a l,

2009a). Regardless o f chemical differences between wood species, desirable chemicals are

produced under the same heating regime.

1.1.3 Chemical characteristic of wood before and after heating

1.1.3.1 Wood extractives and their chemistry

Extractives are chemicals in wood that can be removed using solvents. They mainly consist

of fat, fatty acids, fatty alcohols, phenols, terpenes, steroids, resin acids, rosins, waxes and

other minor organic compounds. Tannins and inorganic salts and certain carbohydrates can

be extracted from wood with water. Most of the extractives exist in heartwood o f both

softwoods and hardwoods. However, softwoods have a higher content of extractives

(Sjostrom, 1981). Most of the extractives may not be essential for plant growth but they play

an important role in defending against wood decay fungi, insects and biological damage

(Céspedes et a l, 2006; Zwenger and Basu, 2008). Three major groups of wood chemicals

are aliphatic compounds, terpenes and terpenoids, and phenolic compounds.

1.1.3.2 Aliphatic compounds

Wood resin contains a large variety o f aliphatic compounds such as alkanes, fatty acids, fatty

alcohol, fats and waxes (Table 1.1). Fatty acids are the major components of parenchyma

resin in both hardwoods and softwoods. They are either saturated or unsaturated. Linoleic

acid and oleic acids are common representatives o f fatty acids. The most important esters are

fats (glycerol esters), usually occurring as triglycerides. Waxes are esters of other alcohols

which are usually aliphatic alcohols or o f terpenoid nature (Sjostrom, 1981).

Table 1.1 Examples o f aliphatic extractives in xylem and bark (Sjostrom, 1981)

Group Structure

n-AIkanes CHs—(CHa)»—CHî n - @-30Fatty alcohols C H ,-<C Fy,-C H ,O H n = 16-22Fatty adds CM r-fCH,),-CDOH n = 10-24Fats CH*-OR R, R', arid R"

(glycerol esters) 1 can be fattyCH— OR' acid residues

talkyl--C(>4CHf-OR* or hydrogen

(mono-, di-, and triglycerides)

Waxes RO—(CHjj),—GHj: R is fatty acid(estere of other alcohols) RO—-sterol

RO—terpene alcoholresidue

Subefln Characteristic; n = 18-28(polyestolides) 1— O — (CHj)n---CO — 1

1.1.3.3 Terpenes and terpenoidsThe constituents of both resin acids and volatile oils are o f a terpenoid nature. The volatile

oil o f conifers contains monoterpenes and their hydroxy derivatives (Figure 1.1). Pinene is

the most common monoterpene in softwood conifers. The resin acids present in the oleoresin

o f coniferous woods are derivatives o f tricyclic diterpenes (Figure 1.2).

OR

Figure 1.1 Examples o f monoterepenes and their derivatives in softwoodl,a-pinene; 2, P-pinene;3, 3-carene; 4, camphene; 5, bomeol; 6, limonene; a-terpineol; 8, dipentene (Sjostrom, 1981)

They are mainly found in heartwood and have a free carboxylic acid functional group.

Dehydroabietic acid is one example of a resin acid (Figure 1.2). Triterpenoids exist in

hardwood and closely related sterols occur in softwoods. Sterols also appear as the alcohol

component in waxes. Polyterpenes and their derivatives also exist in some trees and are

known as polyprenols. They contain various functional groups such as carbonyl, carboxyl,

hydroxyl and ester groups (Sjostrom, 1981).

COOH

C O OH

Figure 1.2 Examples o f resin acids.1, pimaric acid; 2, sandaracopimaric acid; 3, isopimaric acid; 4, abietic acid; 5, levopimaric acid; 6, palustric acid; 7, neoabietic acid; 8, dehydroabietic acid (Sjostrom, 1981)

1.1.3.4 Phenolic extractives

Heartwoods usually contains a large variety o f phenolic substances (Figure 1.3). They have

fungicidal properties and therefore protect the tree against microbiological attack. Phenolic

constituents of wood can be divided into five different groups; hydrolyzable tannins,

flavonoids, lignans, stilbene derivatives and tropolones. Although the phenolic substances

are concentrated in heartwood, only small amounts exist in xylem (Sjostrom, 1981).

OH

4

HO,.OH

OHOH

6OH

5

Figure 1.3 Exam ples o f phenolic extractives.1, gallic acid; 2, ellagic acid; 3, chrysin, 4, taxifolin; 5, catechin; 6, genistein (Sjostrom , 1981 ).

1.1.3.5 Wood structural components

Cellulose, hemicellulose and lignin are structural components o f wood and together they are

regarded as holocellulose. About 40-50% of the wood consists of cellulose which is a linear

polymer chain containing 7,000-15,000 monomeric units of y?-D-glucopyranose, linked by

glycosidic bonds. A group of cellulose chains held together by hydrogen bonding between

their hydroxyl groups is called a microfibril (Figure 1.4).

Cell wall

FibrilPlant cell

MicrofibrilH OH CHjOHH OH CHjOH H OH CH.OH

CHjOH H OH CH,OH H OH CH,OH

C ellulose

Figure 1.4 A irangem ents o f fibrils, micro fibrils and cellulose in cell walls

(Botany Resource Library, 1998)

Hemicellulose is a polysaccharide composed o f monomeric units that are highly branched

and held together by glycoside bonds (Figure 1.5). Each chain consists o f 100 to 400

monomeric units. Glucose and mannose or glucose and xylose are the sugar units o f the main

chain. High numbers of side branches make the hydroxyl groups easily accessible and

moisture in wood is commonly bonded to hemicellulose.

q A HO0 P

GlucomanttaftOH ■ OH

RO"

OH

PHIHO-

Figure 1.5 The most important hem icelluloses in softwoods; xylan and glucomannan (D u ttae /a /., 2012)

Lignin is a three-dimensional network polymer o f wood which consists o f three different

phenol units linked together (Figure 1.6). Lignin comprises 15-25% of the dry weight of

woody plants and is the most complex polymer o f wood because o f the random coupling of

different units. Lignin provides mechanical support to bind plant fibres together and is

known as wood glue.

Figure 1.6 Structure o f softwood lignin (Brunow et al., 1998)

1.1.4 Changes in therm ally modified wood

1.1.4.1 Hydrolysis of wood structu ra l components as a result of heating

The basic characteristics of thermally modified wood have been described by many

researchers. These changes are temperature related. According to Manninen et al., below

40°C, the changes that occur are mainly physical, such as reducing wood moisture and

emission of volatile components. Extractives tend to be more susceptible to heat as they

occur in resin canals and tracheid rays from which they can evaporate and they are less

bonded with structural wood components (Manninen et a l, 2002). As temperature increases,

the changes in chemical composition of wood take place. These changes are due to

degradation of both extractable and structural compounds. Results of many research studies

show that at temperatures above 100°C, all the original extractives have mostly disappeared

and as the temperature reaches above 130°C, structural components start to change and new

chemicals start to appear at certain temperatures. The new chemical properties may result

from changing the ratio between different structural components in the wood (Gonzalez-Pena

efa/.,2009).

Cellulose is the most embedded component in wood and therefore more resistant to

hydrolysis than hemicellulose and lignin (Sjostrom, 1981). However, crystalline cellulose

increases due to the degradation o f amorphous cellulose which is hardly affected by heat

below temperatures of 300°C. The chemical modification starts with the decomposition o f

hemicellulose which is bonded to the surfaces and between cellulose microfibrils when

temperatures reach 180-200°C. This is followed by deacetylation and finally

depolymerisation catalysed by the induced acetic acid. As a result, small organic compounds

with low molecular mass are induced. Lignin degradation starts at around 180-200°C and the

formation of aldehydes occurs because of the decrease in the content o f hydroxyl groups.

Homolytic cleavage o f y5-ether linkages in lignin is affected by heat treatment. Also

formation of condensed structures occurs as methoxyl content is reduced (Tjeerdsma and

Militz, 2005). Possible cross links between lignin and polysaccharides have been reported as

a result o f heating at relatively low temperatures (Gonzalez-Pena et a l, 2009; Niemz et a l,

2010). In addition, the condensation of induced low molecular compounds leads to the

formation of chromophores which are the cause of change in the colour of heated wood

(Beyer et a l, 2006; Sundqvist, 2004).

Pa/'fl-Quinones Stilbenes

■CHj

ort/io-Quinones phenols coniferaldehyde

Figure 1.7 Examples o f chromophores in w ood according to Sundqvist, 2004.(Ri to Re; H or continuing organic chains)

1.1.4.2 Colour change

Colour changes of the wood develop at around 70°C and not only depend on the temperature

but also on the wood species. Softwood becomes slightly darker while hardwood becomes

significantly darker when exposed to heat. The colour change is due to compounds produced

as a result of hydrolysis of carbohydrates (Fengel and Wegener, 1989) and their possible

reactions with other components in the wood. Research on colour change in wood shows that

resistance to biodégradation after heating is dependent on the degree o f change in colour,

which becomes darker with increasing temperature. It has been found that formation of

chromophores such as coniferaldehyde which is a lignin degradation product is a possible

cause of the change of colour in wood (Sundqvist, 2002, 2004).

1.1.4.3 Formation of acid

Wood is naturally acidic, with a pH o f 3 to 6. Extractives such as acidic phenols, low

molecular weight acids and fatty acids give wood its acidic properties. Upon heating of

wood, the formation of further acidic compounds occurs. Acetic acid and formic acid have

been found in emissions from the drying stage of radiata pine at 100°C (MacDonald et a i,

2002). Acetic acid is the predominant acid produced when the temperature is raised to

150°C. Acetic acid, itself, will further accelerate the hydrolysis rate o f wood polysaccharides

(Sundqvist, 2004). A change in quantity and type o f resin acid content of softwoods is

reported by several researchers. Unsaturated resin acids such as dehydroabietic acid are

shown to increase while more saturated forms such as pimaric, linoleic and abietic acids are

decreased (Micales et a i, 1994).

1.1.5 Novel applications for heat treated wood

The market for animal bedding materials from the UK total wood machine waste usage is

estimated to be approximately 400,000 tons of recycled materials, around 56% of the total

(Fletcher et a l, 2010). In general, softwoods are preferred as they do not support bacterial

growth as well as hardwoods and, for example, pine shavings are known to exhibit

antimicrobial activity against food-borne pathogens such as Salmonella and E. coli when

used as animal bedding materials. However, even when natural inhibitors are present in

wood, controlling microbial activity can still be difficult and highlights the need to

investigate how heat treatment could enhance the antimicrobial characteristics in wood-based

bedding materials or mulches.

For this reason the potential beneficial effects o f heat treated wood to be used in two very

different woodehip based systems are considered:

• Poultry rearing systems where food-borne pathogens, primarily Salmonella, must be

controlled as part o f an integrated poultry litter management.

• In woodehip mulches as part of a control strategy directed against the recently arrived

tree pathogen Phytophthora ramorum.

1.2 Background to poultry production and its economic significance

Poultry production is of substantial importance to worldwide economy and agriculture. The

increasing demand for poultry products has resulted in the expansion of animal production

systems in order to meet the protein needs of the human population. The annual poultry meat

production in the U.S., the world's largest poultry producer, totals over 43 billion pounds

(USDA, 2012a). In this 20 billion-dollar industry, circa 65% of the production consists of

broiler meat whilst turkey meat accounts for most of the remainder (USDA, 2012b). Every

year the UK poultry meat industry rears over 850 million chickens resulting in around 1.5

million tonnes of chicken meat. The average poultry meat consumption in the UK is 31kg

per person per year, with a total consumption o f circa 1.9 million tonnes (FSA, 2011). In

2010, the UK poultry production sector comprised 29% breeding and laying fowls, 63% table

chickens and 8% of other poultry including ducks, geese, and turkeys. The broiler share of

poultry meat production increased to a high of 1.32 thousand tonnes or 84% of total

production in 2010 with a value exceeding £2 billion. The versatility o f poultry meat,

particularly chicken, together with its perception as a healthy meat option, has increased the

importance o f poultry in western diets (Crane et a l, 2010).

10

1.2 Heath risks associated with poultry

Despite its economic benefits, intensive poultry production is associated with several major

health issues for humans, food animals and the environment.

1.2.1 Salmonellosis and public health

Bacteria belonging to the species Salmonella are the leading cause of intestinal diseases in

humans and animals worldwide (Castiglioni Tessari et a l, 2012). Estimated costs o f medical

expenses, sick leave and loss of productivity related to the high incidence of salmonellosis in

the US range from 1.3 to 4.0 billion US dollars a year (Taitt et a l, 2004). The annual report

on zoonoses and food-home outbreaks in the European Union reported that Salmonella

accounted for 99,020 reported human food poisoning cases in 2010. Salmonella was found

most often in chicken and turkey meat (EPSA, 2010a, b). In 2009, around 26% of

Salmonella infections reported in the UK were associated with consumption of poultry meat.

In the UK it is estimated that each year approximately one million people suffer from a food-

home disease, accounting for 20,000 hospitalization cases and 500 deaths. The health costs

are likely to be in the region of £1.5 billion (FSA, 2011). In developing countries.

Salmonella is the main cause o f food home disease outbreaks (Majowicz et a l, 2010). It

causes severe intestinal infection in humans accompanied by diarrhoea, fever and abdominal

cramps, symptoms may last for a week or more. In some instances, affected persons later

develop joint pains and arthritis. People of all ages are susceptible but a higher incidence is

reported for infants. Infection may spread to the blood stream, the bone marrow or the

meningeal linings of the brain in people with deficient immune systems causing severe or

fatal illnesses (Tauxe, 1996).

The causal agent

Salmonella was first discovered by Theobald Smith in 1885 and named in honour o f his

director, Daniel Elmer Salmon. Species of the genus Salmonella are characterised as rod­

shaped, non-spore-forming, flagellated. Gram negative bacteria belong to the

Enterobacteriaceae family. There are more than 2,400 different serovars of the bacteria that

all are believed to cause illnesses in humans. According to the current system of

nomenclature adopted by Centres for Disease Control and Prevention (CDC), the genus

Salmonella comprises two species. Salmonella enterica and Salmonella bangori, which each

include several serotypes (Brenner et a l, 2000). Salmonella enterica is divided into six

11

different subspecies. The CDC system uses names for the serotypes of subspecies I, for

example serotypes Enteritidis and Typhimurium.

Salmonella Enteritidis (SE) is the most common serovar (43.5%) of Salmonella isolated from

human worldwide (Hendriksen et a l, 2011). Their ability to survive extreme conditions

combined with a wide distribution makes their control very challenging (Santos et a l, 2008;

White et a l, 1997). In the 1980s, the presence of SE in foodstuffs of poultry origin raised

concern about public health when several outbreaks of foodbome diseases occurred in

England. In the US, there were 189 outbreaks caused by SE with 6,604 patients and 43 deaths

between 1985 and 1989 (Castiglioni Tessari et a l, 2012). According to the European Food

Safety Authority, SE is one o f the most important serovars for public health in the European

Union (EFSA, 2010a). Salmonella Enteritidis is also an important pathogen for the layer

industry primarily because of its ability to infect hens and ultimately contaminate egg

contents (Holt, 1995).

1.2.1.1 Broiler production and associated risk factors of Salmonellosis

(1) Broiler production

The broilers sector o f the poultry industry rears chickens for meat. A typical broiler shed is a

large house housing 10,000-30,000 birds at one time. Chicks are transferred from a hatchery

seetion to the shed at one day old and are kept indoors until they reach the desired slaughter

weight which is around 6 weeks (in some cases birds may be reared outside i.e. free range).

Sheds are heated until ehicks are three weeks old and their body heat can keep them warm.

Food and water are provided. The house condition is monitored and temperature is

maintained by ventilation. The house floor is usually covered with a type o f bedding material

for the insulation from the cold and damp which becomes litter after the chick’s placement

(Figure 1.8). Wood shavings are the most common type o f bedding material but peanut

hulls, rice hulls, straw, shredded papers, and sand are also used. Different poultry production

systems use bedding materials with different depth. In intensive broiler production, 5-10 cm

o f bedding material is layered on the floor prior to placement of chicks. Under the EU

regulations, the entire litter is removed from a house after each flock and before new litter is

applied, the poultry shed is disinfeeted and cleaned thoroughly. In the USA litter from a

previous broiler floek is reused for multiple flocks (for up to one year). Broiler production is

the largest sector of agriculture in the UK. Although free range, bam and organic sectors

have seen growth since 1994, they still remain relatively small (Crane et a l, 2010).

12

Figure 1.8 A broiler grow ing house(http://w w w .bigdutchm an.de/en/poultry-production/hom e/pr-section/photos/poultry-grow ing.htm l)

(2) M ajor sources of Salmonella in broiler houses

Consumption o f meat o f poultry origin is known to be responsible for the major outbreaks of

enteric pathogens (EFSA, 2010a). Contamination o f poultry products may occur at any point

along the food chain from production (broiler houses, layers) to food processing

(slaughterhouses) and preparation (kitchen). Fresh broiler chicken meat is known to be a

major risk factor for SE infections (CCFH-Working-Group, 2007). Like other enteric

pathogens, SE can be disseminated to chickens through a variety o f sources in broiler houses.

Chicken’s gastrointestinal tract

O f the many potential sources reported for Salmonella contamination which include feed,

water, dust, insects, rodents, equipment, faeces and litter, the chicken gut is known to be a

main reservoir in poultry facilities. In conventional production systems, newly hatched

chicks in the clean and controlled environment o f a hatchery will be transferred to broiler

sheds without any contact with adult chickens. Therefore, development of gastrointestinal

microflora (Gl) is largely dependent on environmental sources. A balanced G 1-microflora is

essential to out-compete pathogens and protects the host against enteric pathogens. In the

case o f pathogen appearance in the environment, the sterile intestinal tract o f the newly

hatched chick represents an empty ecological niche for the pathogen to occupy. Salmonella

species can survive the acidic environment of chicken gut (with a pH as low as 3.3 or less) by

an adaption process called the acid tolerance response (ATR) (Foster and Hall, 1990). The

process requires the synthesis o f several acid shock proteins (ASPs) which induces pH

homeostasis and protein repair systems (Foster, 2001). Salmonellae multiply and colonize

the intestinal tract of chicks. Salmonella Enteritidis, like other serovars, causes only a

localized gastro enteric infection in birds (Cogan and Humphrey, 2003). Poultry may carry

different serovars of the pathogen and show slight or no symptoms. Salmonellae can survive

in the gastrointestinal tract of carcasses after slaughter, spreading the contamination into the

processing plant. This can lead to contamination of the whole slaughter line and has the

potential for contamination o f other foods and food preparation utensils. Furthermore, by

law, the detection o f the important serotypes of Salmonella including S. Enteritidis and S.

Typhimurium in early points during production and processing may result in destruction of

the whole flock or slaughter line in some countries. This leads to loss of financial returns for

the poultry producers. Therefore, primary control measures and strategies are established to

control or reduce the pathogen growth in the chickens gut. These include interventions that

aim to assist the development o f a balanced Gl-microflora in chickens growing under

conventional production systems.

Chicken manure

During the growth period, litter is a potential source of Salmonella contamination (Davies,

2005). Enteric pathogens o f the poultry gut are excreted into the litter with manure and

faeces. As chickens continue to live in contact with the litter, it will be seeded with higher

levels o f microbes in the chicken manure as they grow. Outside o f the chicken gut.

Salmonella survival and proliferation is dependent on the temperature and moisture o f the

surrounding environment. In intensive production with a large numbers of birds, litter has an

enormous potential o f becoming reservoirs o f Salmonella.

1.2.1.2 Significance of litter moisture on Salmonella survival

The effect o f high moisture levels in litter can have a dramatic effect on the growth and

spread of Salmonella within the house. Poorly managed litter also has the potential to become

the main source and route o f Salmonella transmission. It can spread the contamination by

sticking to the chicken bodies and carrying the contamination to the processing plants. The

feathers and skin, crop and cloaca o f birds are often highly contaminated (Kotula and Pandya,

1995). The results of extensive research by the United States Department of Agriculture

(USDA) shows that up to 75% of birds in Salmonella positive sites carry the pathogen on

their feathers and feet to processing plants while live birds were only infected at a rate of 1 0 -

15% (Marsh, 1999b). Transmission of Salmonella between (vertical) and among (horizontal)

the flocks is also facilitated by contaminated litter, particularly when litter is reused for

14

raising several flocks. It is reported that 4% of litter is consumed by boilers during grow-out

which is associated with a higher incidence o f Salmonella (Payne and Watkins, 2005). As

chickens contaminated with Salmonella (with the exception o f host restricted serovars) do not

show any symptoms, they can act as vectors, spreading the contamination between flocks

undetected. They further contaminate feed and water with Salmonella by direct contact.

Wet litter has also a direct effect on the microbial composition of litter (Dumas et a l, 2011;

Lovanh et al, 2007). Higher microbial diversity is linked with moisture content o f litter over

the length of the broiler house (Lovanh et a l, 2007). High diversity is believed to increase

the risk of emergence of antibiotic resistant strains of food-borne pathogens o f poultry

(Dumas et a l, 2011). Resistant non-pathogenic bacteria on farms can also act as a pool of

transferrable genes (DeFrancesco et a l, 2004) increasing the emergence o f resistant strains of

pathogens causing food-home diseases such as Salmonella (Angulo et a l, 2004).

The numerous potential sources o f Salmonella contamination in poultry systems increase the

risk of the pathogen carriage in livestock and contamination of food products. As a result, the

impacts of Salmonella incidence are added to public health. Furthermore, strict poultry

legislation of zero tolerance for Salmonella leads to flnancial loss for poultry producers and

growers (the destruction of entire Salmonella positive sites after inspection followed by costs

of substantial sanitation of the broilers house and removal and disposal o f contaminated

litter). As a result, reducing pathogen contamination on the farm seems to be receiving more

emphasis and is in need of interventions that effectively deerease further consequences of

primary contamination carried over to the food processing stage.

1.2.1.3 Control of Salmonella in the gastrointestinal tract of growing broilers

(1) Competitive exclusion

Control of Salmonella infection by competitive exclusion (CE) was first achieved by oral

administration of a suspension of gut content o f Salmonella-frQQ adult birds to 2-days-old

chicks (Mead and Barrow, 1990). Exclusion is considered to result from a range o f direct

effects such as the production volatile fatty acids, competition for colonization sites or

indireet effects such as the stimulation of the host immune system (Doyle and Erickson,

2012). CE treatment is prophylactic and is effective under controlled laboratory experiments

when used against 10 ̂cells/chick (Mead and Barrow, 1990). Under commercial conditions,

however, CE success has been variable due to the absence o f efficacy requirements such as

15

Salmonella-fvQQ chicks, good bio security and low-levels o f stress during the first few days of

treatment (Cox and Pavic, 2010; Goren et a i, 1988).

(2) Probiotics

Probiotics are live cultures of non-pathogenic bacteria that are mixed with bird feed or water.

Microorganisms that are used as animal feed probiotics are mainly bacterial strains o f several

genera such a.s Lactobacillus, Enterococcus, Pediococcus and Bacillus spp.(Gaggia et a l,

2010; Vandeplas et a l, 2010). These treatments attempt to manipulate the microbiota within

the gut, whieh can provide protection against colonization by Salmonella in several ways,

including production of antimicrobial substances such as volatile fatty acids, bacteriocins, or

hydrogen peroxide, reduction of the availability of sites for colonization, competition for

limited nutrients, and stimulation of the immune system (Foley et a l, 2011). Probiotics

provide protection against Salmonella, E. coli. Yersinia enterocolitica, and Campylobacter

jejuni (Schneitz, 2005). However, they have limited suecess in the control of Salmonella for

various reasons. As such, their applications are restricted to minimize the risk of emerging

virulent species and invasive serovars and their potential for antibiotic resistance (EFSA,

2 0 1 0 a).

(3) Vaccines

Another control method is use of a vaccine to reduce Salmonella in the gut as well as in the

reproductive organs. The use o f vaccines against poultry pathogens is increasing (Doyle and

Erickson, 2012). Due to public health problems associated with Salmonella Enteritidis and

Salmonella Typhimurium, these serovars are the target of most vaccinations (Foley et a l,

2011). Both live and killed vaccines are commercially available (Russell, 2012). However,

regardless of the type, they do not guarantee complete protection against the pathogens

(Chambers and Lu, 2002; Gast, 2007) but may decrease the infection burden.

(4) Biosecurity measures

Effective control o f Salmonella outside of chicken gut in the primary production level relies

on biosecurity measures to a great extent. Apart from complementary strategies such as

competitive exclusion and probiotics, restricting movements of birds, people and equipment

combined with substantial sanitation, reduces the risk of zoonotic pathogens. There are

different codes of practiee and standard procedures used in different countries highlight the

significanee of biosecurity (White et a l, 1997). Measures generally include pest control,'

preventative measurements for feed and water contamination, transport sanitation, equipment

16

maintenance and staff training to reduce the risk o f Salmonella contamination (FSIS, 2010).

Management of litter condition during the grow-out period is a major part of biosecurity of

broiler systems (The-Environment-Agency, 2013).

1.2.1.4 Litter control measures

Moisture control

Water activity (Aw) of the litter, which is the level of unbound water, affects Salmonella

growth and survival (Russell, 2012). Since Salmonella is only able to grow at water activities

above 0.94, keeping litter dry is necessary to prevent establishment and proliferation. Results

of a research study by Carr and colleagues showed low water activity values (<0.84) were

associated with Salmonella-nQgaA.i\Q flocks (Carr et a l, 1995). Similarly, it was found that

higher Salmonella and Escherichia coli counts were detected in litter samples with Aw

greater than 0.90 and moisture content greater than 35% (De Rezende et a l, 2001).

However, in intensive production, moisture control is a big challenge and is only possible by

implementing a few strategies such as a low salt and fat diet, effeetive management of

watering systems and using bedding materials with high moisture absorbent properties.

Litter pH control

Salmonella spp. grow naturally between pH 6.5 and 7.5 (Chung and Goepfert, 1970). So the

pH of chicken manure is favourable for Salmonella growth. Mineralization o f organic

nitrogen due to microbial activity in the poultry litter leads to ammonia production in the

litter. Ammonia dissolves in litter moisture and converts to ammonium ions in the litter.

This shifts litter pH toward alkaline which provides the optimum growth conditions for litter

mieroorganisms including Salmonella. Lowering the litter pH has been found to be effective

at inhibiting bacterial growth (Choi et a l, 2008; Moore et a l, 1996; Payne et a l, 2002).

Different types of litter amendments are used to reduce ammonia by reducing pH or, numbers

o f ammonia producing bacteria and direct chernical interactions (Terzich et a l, 1998).

Although, litter amendments or treatments have been shown to be generally effective on

bacteria levels, their use against Salmonella has not been as effective. Line found that

aluminium sulphate and sodium bisulphate reduced Campylobacter colonization frequency

and population in the ceca but not Salmonella colonization (Line, 2002). The greatest

reduction in Salmonella was found when the pH was 4 or less, and showed no significant

reduction at pH of 7 and 9 (Payne et a l, 2007). In a previous study it was found that

aluminium sulphate and sodium bisulphate significantly decreased Campylobacter

colonization in broiler chickens but had no effect on Salmonella numbers (Line and Bailey,

17

2006). Pope and Cherry found no significant effect on Salmonella populations in broiler

litter when using commereially available sodium bisulphate, even though a pH of 1.2 was

achieved (Pope and Cherry, 2000). In another study high levels of both granulated sulphuric

acid and sodium bisulphate (45.4 kg 92.9 m^), which were used in various application levels

on used litter, were needed to obtain low pH for significantly reducing Salmonella

concentrations (Payne et a l, 2007).

Bedding materials

Effect of bedding materials on composition and development o f gut microbiota illustrates

their important role in broiler health and performanee (Garrido et a l, 2004; Ivanov, 2001;

Line, 2002; Torok et a l, 2009). Results of a study by Torok et al. (2009) showed the choice

of litter materials significantly influence caecal microbiota composition and as a consequence

live weight and feed intake. They investigated the effect of bedding type on gut microbiota

composition and broiler performance. Seven types of bedding, softwood and hardwood

sawdust, softwood shavings, shredded paper, chopped straw, rice hulls and refused tea were

tested. Aktan and Sagdic also found that litter type affeets the composition and development

of the microbiota during bird placement (Aktan and Sagdic, 2004). However, others found

microbiota in the gut o f broiler chickens raised on wood shavings and straw developed

regardless of litter type with a slight difference in the first 4 weeks (Fries et a l, 2005),

although, total aerobic bacterial counts were found to be lower for wood shavings than for

straw. Additionally, studies in respect of the effeet o f litter treatment also shows that litter

acidifieation can alter gut and the intestinal flora of broiler chickens and lowers the frequency

of Campylobacter and C. perfrigens, Enterococcus spp. and Lactobacillus spp., (Garrido et

a l, 2004; Line, 2002). Results of these studies illustrate the significance o f chemical

characteristics of bedding materials and their inhibitory effect on microbial activity o f broiler

litter. This potential can be used to effectively control Salmonella during grow-out. The

inhibitory characteristics of bedding materials that could be used to advantage include their

ability to absorb moisture, to effectively control microbial activity o f litter and reduce the

assoeiated health impacts.

1.2.2 Ammonia emissions

1.2.2.1 Animal welfare and human health

A further health risk to human and bird welfare in broiler systems is ammonia emission from

manure. Health and welfare problems associated with high ammonia concentrations in

poultry houses include damage to the respiratory tract o f chickens, increased susceptibility to

18

Newcastle disease, incidence o f airsacculitis,, increased Mycoplasma gallisepticum, and

incidence o f keratoconjunctivitis (Moore et a l, 1995). Adequate ventilation is usually

allowed to exhaust ammonia into the atmosphere. Although ventilation will improve the air

quality of in-house environment, it does not reduce the amount of ammonia in the litter,

leaving birds vulnerable to the negative effect o f high pH on their bodies in contact with the

litter. Skin conditions o f poultry such as breast blisters, ulcers, hulk and foot pad bums are

caused by the high ammonia content of poultry litter. Ulcers can act as gateway for other

infections caused by E. coli and Clostridium perfringens, such as necrotic dermatitis and

scabby hip syndrome, which make broilers suffer from painful inflammations in joints and

under the skin. High ammonia concentrations (20 ppm and above) in poultry houses also

reduees animal growth, feed efficiency, and egg production (Nahm, 2005).

The effects of high ammonia concentrations in poultry facilities on human health are o f great

concern (Moore et a l, 1995). Upper respiratory ailments and eye irritations o f farm workers

are reported to be caused by exposure to concentrations above 25 ppm depending on the time

of exposure and serious health risks are involved at high concentrations of 50-110 ppm

ammonia (Donham, 1996; Ritz et a l, 2009). In addition, ammonia acts as a precursor in the

formation o f ammonium nitrate and ammonium sulphate in the atmosphere which are major

causes of premature mortality, asthma attack and chronic bronchitis (Ritz et a l, 2004;

Tasistro et a l, 2007). By law, the ammonia concentration in broiler houses should not

exeeed 50 ppm as it poses health risks to human operators of poultry (Miles et a l, 2004).

1.2.2.2 Environmental impacts of ammonia emission from broiler houses

Common strategies for reducing the ammonia concentration in poultry units generally aim to

improve the air quality o f the in-house environment rather than inhibiting the formation of

ammonia. Extracting the excessive level of ammonia into the atmosphere has become an

important environmental issue. It has been shown to play an important role in acid rain and

the dominant source of ammonia in Europe is livestock waste (Apsimon et a l, 1987; Moore

et a l, 1996). Ammonia is also a major cause of soil and surface-water acidification, nitrogen

enrichment and aerosol formation when litter is used as a fertilizer. With inereasing growth

of the poultry industry, environmental impacts of ammonia emissions and their effect on air

quality is likely to increase. Strict emission regulations are enforced by environmental

authorities known as Integrated Pollution Prevention and Control Directive (IPPC). In

practical terms, the implementation o f IPPC means that farms that have more than 40,000

birds must ( 1 ) minimise emissions of ammonia, (2 ) reduce associated odour from poultry

houses and (3) have a manure management plan in place. Also, IPPC rules are constantly

being tightened, forcing existing farmers to adjust in the future by implementing new

technologies enabling them to comply with tougher IPPC guidelines (The-Environment-

Agency, 2006). In July 2010, the European Committee proposal to amend the IPPC required

a reduction in the size threshold of poultry units from 40,000 to 30,000 laying hens, to 24,000

for ducks and 11,500 for turkeys. The directive became law in November 2010 (NFU-online,

2013). With profit margins already under pressure, to comply with environmental

regulations, reducing primary production o f ammonia in the litter is only possible by reducing

microbial activity within the litter which results in production of ammonia in broiler houses.

1.2.2.3 Production of ammonia in broiler units

A large and diverse population of bacteria, fungi, viruses and protozoa exists in the poultry

litter. Bacillus spp. and Arthrobactev spp. are dominant mineralizing bacteria (Kim and

Patterson, 2003; Ritz et a l, 2004) and some fungal species such as Aspergillus spp. also

mineralize organic forms of nitrogen into ammonia (Cook et a l, 2008). Mineralization of

organic nitrogen naturally takes place due to microbial activity in the poultry litter and leads

to ammonia production (Figure 1.9). Undigested proteins, uric acid and urea are the main

source o f organic nitrogen excreted as manure. Microbial degradation of uric acid in the litter

occurs in two steps; first urea is formed as an intermediate compound. The decomposition

process requires uric acid, water, and oxygen to react, releasing NH 3 and carbon dioxide

(CO2). The water-soluble characteristic of NH 3 allows it to be dissolved in the moisture,

protonating ammonia NH 3 into the non-volatile ammonium ion (NJU^.

NH 3+ H 2 O ± = ^ NH4"^+0 H‘

In broiler units, high stocking density decreases the rate of moisture release from the litter.

However, the respiratory output o f large numbers o f birds especially toward the end o f the

grow-out period increases the relative humidity and temperature of the in-house environment.

Litter absorbs excess water in the atmosphere. High moisture content in conjunction with

high temperatures in the broiler houses facilitates microbial breakdown o f the manure leading

to a higher content of ammonium ions in the litter. Ammonium ions will be converted into

ammonia (NH3), so the pH of the litter shifts toward alkaline and ammonia volatilization

increases. The inactive life style o f chickens (as a reflection of high stocking density) means

longer exposure o f their body to direct contact with litter. It is therefore necessary to control

2 0

the condition of the litter during the grow-out period. Moisture and pH of the litter are the

main important factors in controlling ammonia production in broiler units.

1.2.2.4 Ammonia production control and litter management

(1) Moisture control

Best management practice guidelines are issued to help farmers and producers to manage

litter efficiently by the Environmental Agency (The-Environment-Agency, 2013). According

to BMP (Environmental Management Plans) guideline for intensive farming, maintaining

moisture content o f 20-25% is recommended to avoid odour problems of wet litter. A

substantial depth o f litter o f at least 10-15 cm together with managing water leaks or spillage

due to faulty drinkers or leaks from walls and ceilings will control the moisture of the litter to

optimal level.

Free air stream

g.»

IN H g (aq) + H

Biomass

N H 4 ̂(adsorbed)

L it te r solidM oistu re

layer

Convection mass transfer

Partitioning betw een aqueous and gaseous phase ammonia

Chemistry o f ammonia in aqueous solution

Ammonia generation

Partitioning betw een solid and aqueous phase ammonia

Figure 1.9 Illustration o f processes related to am m onia em issions from litter based manure (Liu et a i , 2007)

Absorbent amendments are also sometimes recommended to reduce extra water in the litter,

although their use is limited by cost and their variable seasonal effects. Ventilation is

traditionally a common practice to lower ammonia concentration o f poultry units. Air

scrubbers and bio filters are other technologies that aim to clean air by exhaustion. However,

their practical applications are limited by cost and size restrains o f conventional poultry

houses (Ritz et a i, 2004). Reducing nitrogen content o f manure based on diet manipulation

such as reducing protein uptake and amino acid requirements also results in lower ammonia

production. However, as litter ages, moisture control becomes very challenging for the

21

poultry producers, particularly in re-used production systems. Furthermore, to maintain the

temperatures of the in-house environment during cold seasons, ventilation is reduced.

Therefore, litter amendments are commonly used to reduce ammonia volatilization by

lowering the pH of broiler litter.

(2) Litter amendments

Litter amendments are chemical treatments that are used to reduce ammonia emissions in

broiler houses. Different types of litter amendments are used to reduce ammonia by

reduction in litter pH, numbers o f ammonia producing bacteria and direct chemical

interactions (Terzich et a l, 1998). Currently, the most effective products have been found to

be chemicals that lower the pH of the litter. When litter pH is at levels o f less than 7.0,

ammonia production is insignificant. But at pH levels of greater than 7.0, ammonia

formation increases and when greater than 8 , it provides optimum growth condition for

Bacillus pasteurii, one of the primary uricolytic bacteria that facilitates NHg production

(Bacharach and Evans, 1957; Carr et a l, 1990; Reece et a l, 1979; Scheffer, 1965).

Typically, the pH of poultry manure as excreted and the pH of litter are between 7.5 and 8.5.

Lowering the litter pH has been found to be effective at inhibiting bacterial growth (Choi et al,

2008; Moore et a l, 1996; Payne et a l, 2002).

Choi et al, (2008) found using aluminium sulphate, aluminium chloride, and ferro sulphate

significantly reduced total aerobes and Gram negative bacteria in all three treatments. They

studied the effect of chemical treatments on pH and bacteria level o f litter treated with alum,

AICI3 and FeS0 4 at the same concentration rate ( 8 g 100 g litter) for 3 weeks. All treatments

reduced numbers of aerobic bacteria by 22 to 87% and Gram negatives by 63-99% of

untreated litter. Aluminium sulphate has been also shown to be able to either eliminate or

reduce aerobic bacteria levels in the litter (Line and Bailey, 2006). Others have reported that

acidic treatments reduce the survival and reproduction of the total populations o f different

microorganisms in the litter.(Choi et a l, 2008; Moore, 2003). However, litter amendments

are more affective at controlling pH when they are used above the manufacture’s

recommended rate (Wheeler et a l, 2008). This has increased concern about emerging

virulent strains of bacteria as a result of being exposed to high levels of chemicals (Payne and

Watkins, 2005). Furthermore, the effect of litter amendments lasts only 10-14 days while the

broilers are susceptible to ammonia until they become 21 days old (Wheeler et a l, 2008).

2 2

(3) Characteristics of wood materials as animal bedding materials

Physical parameters

Bedding materials are traditionally used to provide a dry and warm environment between

birds and to protect them from the cold floor. Different types of bedding materials such as

saw dust, rice hulls, straw, shredded papers and wood shavings are commonly available as a

by-products of other industries (Ritz et a l, 2009). The effect o f bedding materials on litter

condition and broiler performance has been extensively studied (Atencio et a l, 2010; Bilgili

et a l, 1999; Carlile, 1984; Hess et a l, 2010; Macklin et a l, 2005; Malone et a l, 1983;

Maitland, 1985; Oliveira et a l, 1974; Willis et a l, 1987). From these studies it can be

concluded that the ability of bedding materials to absorb moisture effectively reduces health

risks caused by high microbial activity of the litter such as ammonia emissions. Microbial

composition of poultry litter has been shown to vary due to the physical parameters of

bedding materials such as moisture content, pH and temperature (Aktan and Sagdic, 2004;

Lovanh et a l, 2007). However, a study by Miles (2011) comparing organic and inorganic

materials shows water absorbance is not a reliable indicator for good bedding materials. In

this study, NH 3 content o f pine, rice husk, sand and vermiculate in two different level of

moisture were compared. Results showed that at the original moisture content, sand and

vermiculate produced the most NH 3 (sand: 5.3 mg, vermiculate: 9.1 mg) and commercial

wood shavings and rice husk produced the least (wood shavings: 0.9 mg, rice husk: 0.26 mg)

while vermiculate was the more absorbent compared to wood materials (Miles et a l, 2011a).

This has drawn the attention of the research to chemical characteristics o f bedding materials

and its effect on microbial population of chicken litter.

Chemical characteristics

Several studies have found that the reduction of ammonia in the litter is linked with lower

microbial activity due to inhibiting effects of bedding materials rather than physical

parameters (Atapattu et a l, 2008; Duqueza, 1996; Lien et a l, 1998; Miles et a l, 2011b).

Duqueza (1996) suggests that lower ammonia emissions from wood shavings are due to

higher lignin content. Lien (1998) also found that using wood shavings reduced ammonia

compared with peanut hulls. In another study by Atapattu (2008), the NH 3 emission o f three

bedding types, refused tea, sawdust and paddy husk were studied. The lowest emission was

recorded from refused tea (13 mg kg'^ per hour) compared with sawdust (54 mg kg" ̂ per

hour) and paddy husk (44 mg kg’* per hour) after 42 days. The authors suggest that the

polyphenol content of refused tea lowers microbial activity and ammonia concentration.

23

These results are in disagreement with those of Duqueza because paddy husk, with higher

lignin content, had higher ammonia emission compared to refused tea (44 vs. 13 mg kg'^ per

hour). Nevertheless, wood materials are known as the most effective and commonly used

bedding materials because o f their good moisture absorption properties (Embury, 2004; Miles

a/., 2 0 1 1 b).

1.2.2.5 Limitation in using bedding materials and alternative approaches

The efficiency o f wood materials is supported by results o f many research studies on their

effects on reducing ammonia emissions and their major impacts on broiler health and

reduction in mortality and skin conditions of broilers (Ferrante et a l, 2006; Miles et a l,

2011b). However, wood materials are likely to become the habitat of different

microorganisms such as pathogenic bacteria and mould because of their porous structure. In

other industries, where hygiene of wood is critical, such as in food packaging, wood materials

are sanitised using different techniques such as washing, radiation, steam and dry heating

before use. In contrast, wood materials used to raise poultry are usually the unprocessed end

products of other industries such as the timber industry (Fletcher et a l, 2010). New bedding

materials are shown to be a potential source o f Salmonella at the time o f placement o f new

flocks and untreated materials also increase the risk of contamination with Salmonella during

storage (Fries et a l, 2005; Volkova et a l, 2009).

Although hygiene of bedding materials and their storage condition is suggested to influence

litter condition during grow-out, the effect o f potential treatments such as heating which can

be done to improve the quality of wood materials as animal bedding has not been

investigated. Research on efficacy or potential applications o f wood materials has been

focused on improving the life duration and resistance toward wood decay fungi. Therefore,

most of the existing developments are based on the need in the market for commercially

profitable products and their applications although, the market for animal bedding materials

in the UK, is the second biggest market for recycling the wood materials o f other industries.

Little has been done to develop heating techniques that improve the hygienic condition of

bedding materials before use (Fletcher et a l, 2010). Heating also has the potential of

introducing valuable levels of chemicals with antimicrobial properties, thereby reducing

zoonoses diseases of poultry as well as improving welfare of animals and humans and air

quality by reducing ammonia emission.

24

1.3 Background to the im pact and biology oi Phytophthora ramorum

Sudden Oak Death is a relatively new disease caused by the invasive plant pathogen

Phytophthora ramorum. In the mid 1990’s the disease was noticed killing tanoak trees

{Lithocarpus densijlorus) over a matter of weeks in some forests of California in the USA

(Rizzo et a l, 2002). Since then, a similar phenomenon has been observed to occur in

fourteen counties from Monterey County in the south to Humboldt County in the north

(Kliejunas, 2010). The mysterious causal agent was unknown until it was isolated and

identified as a species of Phytophthora. It then transpired that it was the same species, named

P. ramorum in 2001 (Werres et a l, 2001), that had been isolated from nursery plants of

rhododendron and viburnum in Germany and Netherland in the mid 1990s.

P. ramorum has since been discovered mainly on nursery stock in a number of European

countries including Germany, Netherlands, Belgium, the Czech Republic, France, Ireland,

Italy, Spain, Slovenia, Sweden, Denmark, Switzerland, Norway and Serbia. The first case of

P. ramorum in the UK was confirmed in a nursery in April 2002 on Viburnum tinus (Lane et

al, 2003). At the time of this discovery, the disease was causing damage to the forests and

wildland in the USA but it was only confirmed in a number o f nurseries in Europe. In 2001,

however, the disease was found on rhododendron in a Californian nursery, followed by the

first report of an infected tree outside the United States in November 2003 when P. ramorum

was isolated from a mature southern red oak tree {Quercus falcata) in the UK (Brasier et a l ,

2004; Goheen et a l, 2002). Since 2003, and despite quarantine efforts and emergency

regulations put in place in both Europe and the USA, the disease has spread fast and infected

a broad range of plant species (Frankel, 2008). In 2004, 176 nurseries were reported positive

after inspection in 21 states of the United States, apparently as a result o f interstate shipments

out of infested nurseries in California (Garbelotto and Rizzo, 2005; Jones, 2006). In Oregon,

approximately, 971 ha of the United State forest has been subject to eradication o f all infected

and nearby host plants between 2001 and 2008 (Kanaskie, 2010).

In 2007, several tree species with symptoms of bleeding bark cankers were identified in the

United Kingdom in Cornwall, mainly beech, but with some other species showing symptoms

of foliar infection and dieback, for example ash and holm oak (Sansford, 2007). In 2009,

Japanese larch was found with both stem and foliar symptoms in southwest England (Webber

et a l, 2010 ). In 2010, infection of larch became widespread with the disease spreading to

south Wales, where it has killed large numbers o f mature larch trees. (Figure 1.10)

25

a - ê i ^%» ■- -«Ÿ* ' ,

i ^ f K k mr f

3r} .;:>,'V4!SS Î ■s

Figure 1.10 Sym ptom s o f sudden oak disease in woodland and forest.Left, dead and dying tanoak in California. July 2010. Right, similarly afflicted larch forests south w est England, May 2010 (Source: w w w .nature.com /.../n7308/fig ).

1.3.1 The causal agent of Sudden O ak Death: Phytophthora ramorum

The pathogen is a member of the genus Phytophthora, and many o f its species pose a serious

threat to a range o f agricultural crops. Phythophthora belongs to the Oomyctes or water

moulds which are diploid eukaryotic microorganisms. Oomycetes were traditionally

classified as fungi but are now assigned to the Kingdom Chromista. Most known

Phytophthora species are soil-borne, root-infecting plant pathogens (Erwin and Ribeiro,

1966) but P. ramorum is an exception in that it is an air-borne species. It produces two types

of spores: chlamydospores and zoospores and the latter are contained in sporangia. Both are

vital for the survival and successful, spread o f the disease (Figure 5.2). Chlamydospores are

resistant to adverse environmental conditions and are known as resting spores, allowing the

pathogen to overwinter or persist during adverse environmental conditions. Sporangia are

asexual reproductive structures that contain zoospores and both are able to germinate and

infect plants directly. Zoospores have flagella enabling them to swim in water films

(Judelson and Blanco, 2005). Phytophthora ramorum is heterothallic. needing two mating

types, Al and A2, to produce oospores. Oospores are sexual spores which, in case o f P.

ramorum. have not yet been found in nature (Grunwald et al.. 2012)

26

Figure 1.11 Sporulation o f Phytophthora ramorum.Hyphae grow through lea f tissue and produce sporangia and chlam ydospores (Source: w w w .suddenoakdeath.org/w p-content/gallery/p-r)

Until 2012, three lineages o f P. irtmontm were known, termed EUl (European lineage 1),

NAl and NA2 (North American lineages 1&2) (Grunwald et al., 2012). The EUl lineage has

been found in European nurseries and forests and it has caused the current epidemic on

Japanese larch in the UK. There are also reports o f its recovery from US west coast nurseries

and waterways. The NAl is commonly isolated from infected forests in California and on

occasions from US nurseries. The NA2 is recovered mostly from US nurseries and is the

dominant lineage in western Canada (Grunwald et a l, 2012). Recently, a fourth genetically

distinct lineage has been identified, and designated as EU2 and is currently limited to

southwest Scotland and Northern Ireland (Poucke el a l, 2012).

1.3.2 Symptoms caused by Phytophthora ramorum

Infection by Phytophthora ramorum causes two different types o f disease: bleeding cankers

on the trunks and branches o f tree species and dieback and leaf blight of mainly shrubs and

ornamentals. Symptoms of the disease on tree species appear on the lower trunk in the form

of bleeding cankers (Brasier et a l, 2002) (Figure 1.12). When P. ramorum grows into the

bark it destroys the cambium and can eventually girdle the tree. At this stage, older leaves

start to become pale green and in a matter of two to three weeks they turn brown while

remaining attached to the tree. Symptoms on foliar hosts include necrotic spots appearing

along the leaf margins and leaf tips throughout the crown. Spores that are formed on the

infected leaf and shoot tissue (Figure 1.11) of foliar hosts but these can then also infect

leaves and bark of other plant species (Figure 1.12).

27

Figure 1.12 The sym ptom s o f infection by Phytophthora ramorumRhododendron leaves (left) and on the right, bark cracks with black exudates in coast live oak caused by P. ramorum. (Source: http://w w w .forestresearch.gov.uk/fr/IN FD -7B 5K U 2 )

1.3.3 Risk factors of Phytophthora ramorum

1.3.3.1 D istribution

The native origin o f P. ramoimm is unknown, but there is evidence that the pathogen has been

introduced separately into North America and Europe from another undiscovered area. The

distribution o f P. ramorum now includes the United States, where the disease was first seen

in 1995, Canada and several European countries (Brasier, 2003; Grunwald et a l, 2012; Ivors

et a i, 2004; Rizzo et al., 2005).

In the United States, the disease was first reported from forests in California and Oregon.

Since the first disease signs in 1995. and despite control efforts, from 2001 through 2008

approximately 971 ha o f forest has been burnt or cut to prevent further spread. The first

report o f P. ramorum from nurseries was made in 2003, when it was isolated from plant

containers o f rhododendron in Santa Cruse County. In 2004, the number o f positive nurseries

increased markedly due to interstate shipments o f affected plants. In 2009. the US

Department o f Agriculture, Animal and Plant Health Inspection Service reported 11 states

where P. ramorum had been detected.

In Canada. P. ramorum has been limited to nurseries since the first report from potted

rhododendrons in British Columbia in 2003. Eradication treatments have been successful to

control the spread o f f . ramorum and it is not present in the wider environment.

Since the first reports in Germany and Netherlands (Werres et al.. 2001). P. ramorum has

been discovered mainly on nursery stocks in a number o f European countries including

2 8

Germany, the Netherlands, Belgium, the Czech Republic, France, Ireland, Italy, Spain,

Slovenia, Sweden, Denmark, Switzerland, Norway and Serbia. However, in Belgium,

Denmark, France, Germany, Ireland, Luxembourg, the Netherlands, Slovenia, Spain and the

United Kingdom infected plants have been reported outside nurseries, in managed gardens

and woodlands (Sansford et a l , 2009).

In the UK, P. ramorum was first found on a mature oak tree {Quercus falcata) in Sussex in

2003 (Brasier et a l, 2004). It was the first confirmation of a diseased tree outside the United

States. About the same time, infection was also found on several Quercus rubra trees in the

Netherlands (Werres et a l, 2001). Since then, it has been found on a wide range o f plant

species in the UK. By 2007, limited numbers of tree species were found infected with

symptoms of stem canker but foliage and dieback symptoms were mainly reported on

rhododendron, viburnum and camellia. In 2009, Japanese larch trees were found with both

stem and foliage symptoms in Devon, Cornwall, and Summerset in southwest England (FC,

2012; Webber et a l, 2010 ). Until these findings, the majority of woodland infections in the

UK were associated with rhododendron which acts as the sporulating host, producing many

spores which then lead to infection of other species. Interestingly, in the absence of

rhododendron in these newly infected sites, needles of infected Japanese larch were found to

be producing large numbers o f spores, even more than had been previously found to be

produced on rhododendron leaves (FC, 2012). Therefore, felling infected larch trees in

forests was commenced alongside removal o f rhododendron to prevent the production of

spores and thus reduce the risk o f further infections and spread. By mid 2010, P. ramorum

had been confirmed on larch in both England and Wales (Brasier and Webber, 2010). Despite

taking emergency action in cutting diseased trees and putting biosecurity measures in place,

infection has been also found affecting larch in southwest Scotland and Northern Ireland.

1.3.3.2 Host factors

(1) Range of Plant hosts

Phytophthora ramorum infects a wide range o f plant species, including shrubs and

ornamentals, heathland plants and many tree species. To date, more than 150 plant species

are known to be susceptible to P. ramorum worldwide. In the United States, where the

disease was first discovered, the main host is known to be oak {Quercus) but other tree

species are also reported as hosts such as pacific yew, Douglas fir, grand fir and coastal

redwood. However, as a result of the significant damage to oak trees, the disease is well-

29

known as sudden oak death in the United States, where P. ramorum has killed large numbers

of North American native and tanoak trees (Frankel, 2008).

In the UK, more than forty plant species have been found to be susceptible to attack by P.

ramorum including red oak, beech, ash, yew, rhododendron, viburnum, magnolia and

heathers and bilberry (Brasier et a l, 2002; Denman et a l, 2005a; Denman et a l, 2005b;

Denman et a l, 2005c). However, the two native English oak species {Q. robur and Q.

petraea) have been shown to be more resistant and are less affected by P. ramorum than

American native oak species so the disease is known as “ramorum disease” rather than

sudden oak death in the UK. Until 2009, woodland infection was not widespread and most

infections occurred in nurseries. In 2010, infection of Japanese larch became widespread in

some part of the UK. Hybrid and European larch are also infected occasionally. Sitka spruce

is also reported to be infected under high levels of inoculum pressure (Chastagner et a l,

2012).

(2) Role of foliar hosts on disease cycle and development

Foliage hosts play an important role in the dispersal and epidemiology o f ramorum disease as

they are the main source of spores (Rizzo et a l, 2002; Turner, 2007). Tree hosts that are only

susceptible to trunk bleeding canker, such as oak, do not produce spores from infected tissue

(Davidson et a l, 2005a; Garbelotto and Rizzo, 2005). Studies on the disease cycle show that

P. ramorum is reliant on foliar hosts for reproduction and survival (Figure 1.13). California

bay laurel (Umbellularia california) was found to be the significant sporulating host for large

numbers of oak trees infected by P. ramorum in California forests (Kelly and Meentemeyer,

2002; Swiecki and Bernhardt, 2001). The mortality rate in oak trees in California forests was

shown to increase with increase in the number o f bay laurel associated with high levels o f

inoculum (Condeso and Meentemeyer, 2007). Cushman and Meentemeyer suggest there is a

lag effect between infections on bays and appearance of symptoms on oak trees (Cushman

and Meentemeyer, 2006). In Europe, Rhododendron ponticum has been found to be the key

sporulating host for the infection on susceptible tree species. In the UK, Rhododendron

ponticum and Larix kaempferi provide primary inoculum for most stem infections o f tree

hosts (Brasier and Jung, 2006; Brown et a l, 2006; Turner, 2007). Other foliar hosts such as

bilberry, holm oak and sweet chestnut are also suggested as significant sporuator hosts for

ramorum infection in northern Europe (Inman et a l, 2005; Sansford et a l, 2009).

30

A. Sporulation on infected lear es of foliar host

C. Dying crown o f a bole canker host

B. Twigs and leaf infection

D. Necrotic lesion in inner bark under bleeding canker in outer bark

£. Sporulation on fallen leas es

(D Primary- inoculum (sporangia) produce on (D Local intensification o f disease caused by ® The pathogen can persist in soil forinfected leaves can be carried \tia rain splash and air currents, infecting new hosts.

(D Secondary inoculum (sporangia or zoospores) is carried down stems by rainwater to infect lower portions o f the tree, infecting the inner bark and sapwood. resulting in a bleeding canker.

secondary inoculum which is produced in the canopy

® Infected leaves can act as a source o f inoculum even after they fall to the ground

® Sporangia produced on fallen leaves are carried to leaves and lower stem o f shrubs by rain splash

several months presumably as ÇjUainydpsEprçs.

F igu re 1.13 The disease cycle o f P. ramorum

(Parke and Lucas, 2008)

1.3.3.3 Environm ental factors

Temperature and moisture are the two main environmental factors that play important roles in

P. ramorum epidemiology. Phytophthora ramorum is regarded as a eool-temperature

organism which has an optimum growth temperature o f between 18-22°C (Werres et al.,

2001 ). The asexual spores, zoospores and chlamydospores, are produced under a broad range

of temperatures. Laboratory studies show that NA and EU lineage isolates produce sporangia

at temperatures between 10 to 30°C and 6 to 26°C respectively (Englander et a l. 2006).

Similarly, chlamydospore production o f U.S and European isolates is favoured by a

temperature range o f 10 to 28°C and 10 to 26°C respectively depending on the isolate.

However, abundant production o f sporangia and successful germination o f zoospores is

dependent on humidity. The maximum level o f zoospore germination is observed al 100%

humidity at a temperature range o f between 1()°C to 30°C. The presence o f free water in

susceptible plant parts, such as at leaf tips, enhances the infection by zoospores (Garbelotto et

6//., 2003).

1.3.4 Dispersal

Inoculum of Phytophthora ramorum spreads by different means after production on host

tissue. The spread can be by rain splash, in wind-driven rain, and in turbulent air locally

(Davidson et a l, 2002; Davidson et a l, 2005a; Fichtner et a l, 2009; Hansen et a l, 2008).

Long distance dispersal is mainly due to the movement of infected materials by local,

national and international trade of ornamental plants (Davidson and Shaw, 2003) and via

water courses (Davidson et a l, 2005a; Turner, 2007). Human activities also contribute to the

introduction of the pathogen into uninfected areas (Cushman and Meentemeyer, 2006).

Infected plant matter and soil can stick to vehicle tyres, shoes and tools and thus can spread

the disease over great distances (Cushman et a l, 2008; Tjosvold et a l, 2002a; Webber and

Rose, 2008). During 2006, dispersal of the pathogen up to a distance of 50 meters away from

an infected host was reported in the UK, by using water traps to track spores of P. ramorum

(Turner, 2007).

1.3.5 Survival

The potential o f disease caused by P. ramorum is dependent on its survival during

unfavourable conditions. The pathogen is able to survive dry and hot conditions in host

tissue materials. Like some other species of Phytophthora, chlamydospores are the resting

structures in the disease cycle and these are produced abundantly in the leaf tissue o f infected

foliar hosts (see Figure 1.11). Laboratory studies show chlamydospores and zoospores are

also able to survive at least for a month in moist conditions outside host tissue (Davidson et

a l, 2002). Bay laurel leaves are known to be an important niche for the pathogen during hot

summers in California (Rizzo, 2006). Survival in abscised leaves is also significant in the

disease cycle, with the survival in buried leaf litter shown to be much higher than in leaves on

the ground surface (8 8 % vs. 26-66%) (McLaughlin et a l, 2006). Other studies have reported

survival o f P. ramorum in rhododendron leaf tissue buried in potted nursery stock after 150

days (Shishkoff and Tooley, 2004). It was also recovered from potting media and sand over

approximately a year with no significant difference among treatments over time (Shishkoff,

2007). The pathogen can also survive in artificially infected host leaves buried or in the

ground. Turner et al, (2005) reported recovery o f P. ramorum for two winters from an

experiment conducted in the UK when it could be isolated from at least 50% of infected

leaves in all treatments. Survival of P. ramorum in woody materials is also reported. Shelly

et al. (2006) recovered P. ramorum from 1 out of 30 specimens o f tanoak and coast live oak

logs that had been air-dried for 6 months. P. ramorum has also been shown to survive in the

32

exposed wood of trees for up to two years (Brown and Brasier, 2007). Similarly, P. ramorum

survives the summer season in Rhododendron ponticum leaves in the UK.

Phytophthora ramorum \s occasionally isolated from soil during hot summers (Davidson et

a l, 2002; Davidson et a l, 2005a). Failure or decrease in P. ramorum survival in soil is

known to be associated with gradual soil drying during hot summer months (Davidson et a l,

2002; Fichtner et a l, 2007b). Phytophthora ramorum has a high potential to survive in

potting media. In an experiment where sporangia and chlamydospores where artificially

inoculated with the media, up to 6 and 1 2 months survival was observed respectively

(Linderman and Davis, 2006; Linderman et a l, 2006). Survival in potting media and sand is

reported to be longer at 4°C compared with survival at room temperature (Jeffers, 2005). In

the UK, laboratory studies on the effect o f temperature on chlamydospore germination

showed complete inhibition at extreme temperatures (-25°C and 40°C). In a study by Swain

et al. (2006) on the effect o f temperature during composting, no chalamydospores were

recovered after exposure to 55°C for an hour or 40°C for 24 hours with similar results

obtained in another study (Swain et a l, 2006; Turner and Jennings, 2005).

1.3.6 Disease current management

Current disease management involves two main principles: prevention and suppression.

Prevention includes actions taken before the pathogen is introduced such as import control or

inspection. Exclusion and eradication are the primary strategies that are aimed at preventing

the spread of inoculum or to control disease outbreaks.

1.3.6.1 Exclusion

Regulatory actions are implemented worldwide to reduce or eliminate the spread o f P.

ramorum. Different countries have restricted the movement of plants or plant products from

the United States. Sixty-eight countries are listed in the European Union Pest Risk

Assessment for which P. ramorum is part o f their legislation or considered in their Plant

Health Regulation (Sansford et a l, 2009).

The United States has enforced interstate and international plant quarantine measures. In

2001, the first quarantine measures were issued by Oregon State to prohibit the introduction

of P. ramorum from California where the disease was first noticed. Also, in 2002, the

European Union took emergency action by restricting the import of susceptible hosts into

member countries from the United States (2002/757/EC). For the movement of the main

known hosts in the EU (rhododendron, viburnum and camellia), plants must be certified by a

33

system called a Plant Passport which aims to verify that plant materials are inspected and free

o f infection (Frankel, 2008; Rizzo et a l, 2005). Under EU regulations, all member states

must undertake surveys to keep legislation continuously updated for P. ramorum. In the UK,

monitoring of P. ramorum was started after the first reported outbreaks in large managed

gardens in 2003. DEFRA commissioned a research programme for monitoring

contamination in infected areas following eradication actions. Seasonal pathogen survival,

pathogen persistence in water, litter, and soil and inoculum dispersal were all investigated to

inform the pathogen risk analyses in the UK. A range of methods for monitoring P. ramorum

are used which includes leaf baiting followed by traditional culturing methods and quatitative

polymerase chain reaction method (PCR) to detect low levels of inoculum. If infected plants

are found they have to be destroyed, and other phytosanitary measures are applied to prevent

further spread. For primary detection of Phytophthora species in the field, an ELISA kit

known as Lateral Flow Device (LFD) is developed by Forsite Diagnostics Ltd., York, UK.

The kit is easy to use and gives results in a few minutes. LFD kits are widely used for field

diagnosis of Phytophthora species and their results are usually comparable with PCR and

other laboratory techniques.

1.3.6.2 Best management practice

To reduce the adverse effect o f the disease, different measures, methods and practices have

been introduced through experience, extensive research and background knowledge o f the

disease cycle. This information has been distilled into guidelines for preventing or managing

of P. ramorum outbreaks in both woodlands and nurseries including measures to prevent the

spread during pruning, planting and soil handling in regulated areas. The guidelines may

vary between woodlands and nurseries, although applying a combination o f the suggested

measures is usually most effective. A best practice guide for woodland, parks and gardens is

developed and published by DEFRA (Defra, 2008).

1.3.6.3 Eradication

The current strategy to control and reduce the spread o f P. ramorum where it is already

present is aimed at lowering the inoculum levels in the environment by removing infected

sporulating hosts. Although, these efforts are likely to be successful if the disease is notified

shortly after it has been introduced, they can be expensive and costly. In the United States,

2331 hectares of Oregon State lands around the disease centre were subjected to Oregon and

APHIS quarantines (Kliejunas, 2010). The results showed a reduction in the number o f new

infested areas over a few years, reducing the damage to forests. Since 2002, FERA (Food

34

and Environment Research Agency) has launched a programme to clear rhododendron from

woodland and other sites to control ramorum in the UK. The process has been successful at

some sites (Turner, 2007), but not completely at others. In response to the outbreak o f P.

ramorum on Japanese larch, the Forestry Commission felled 600,000 infected larch trees in

2010 which were in private and public sites in south west o f England, compared with more

than 60 ha of rhododendrons cleared from woodlands in 2007 (Brasier and Webber, 2010).

After removal of rhododendron, herbicides are applied on the remaining stumps to prevent

resprouting. Similar actions are taken as emergency requirement against P. ramorum

throughout European Union countries (Aveskamp et a l, 2006; Defra, 2008).

1.3.6.4 Heat treatment

Heat treatment has been examined to assess its potential to reduce the pathogen levels in

infected plant materials. Composting has been shown to be effective at reducing P. ramorum

in green waste (Swain et a l, 2002; Swain et a l, 2006). A temperature of 55°C for 2 weeks

has been demonstrated to be sufficient for complete elimination of P. ramorum from compost

piles. However, the risk o f contamination remains if the composting process is not done

correctly. In addition, the prolonged heat treatment is not practical for some hosts. Steam

heat has been shown to be more effective than dry heat to decontaminate the plant containers

or infested media (Linderman and Davis, 2006). Heat treatment is also used to

decontaminate conifer bark before it can be moved from non-EU countries. An hour at 55°C

is also considered to be sufficient heat treatment to stop viable P. ramorum remaining in

cankered stems and woodchips of affected trees (Shelly et a l, 2006).

1.3.6.5 Chemical treatments

A variety of different fungicides used against other species o f Phytophthora have been tested

on P. ramorum. Many o f these are found to be effective in vitro, and include metalaxyl, Al-

fosetyl, copper sulphate, copper hydroxide and phosphonate (Garbelotto and Rizzo, 2001;

Hardy et a l, 2001). In the USA, the effect of fungicides has been evaluated using different

application methods such as trunk injections, soil drenches, foliar spray and topical

applications (Garbelotto et a l, 2003). Trunk injection of the active ingredient (phosphonate

compounds) was found to be the most effective method for controlling P. ramorum.

Although chemical treatments are found to be effective, the practicality o f their application is

limited to individual high-value trees rather than widescale application in woodlands and

forests. Foliar treatment o f oaks and tanoaks was tested using phosphonate but it was found

to only provide short-term protection accompanied by some phytotoxicity (Garbelotto, 2004;

35

Garbelotto et a l, 2007). Chemical treatment o f irrigation water and soil with compounds

such as hydrogen peroxide and sodium hypocholorite can be effective at decontaminating

water containing P. ramorum propagules (Sansford and Woodhall, 2007). Chemicals

containing copper are also toxic to propagules of P. ramorum (Colburn and Jeffers, 2010). A

variety of different chemicals are used as drenches to control P. ramorum in soil such as

metalaxyl-M, chloropicrin and dazomet (Yakabe and Macdonald, 2008) and can either reduce

the number of propagules or eliminate the pathogen (Turner et a l, 2006; Yakabe and

Macdonald, 2008). However, there is concern over widespread application of metalaxyl-M

as development of emerging resistant isolates of P. ramorum has been detected (Heungens et

a l, 2006; Turner et a l, 2008). There are also concerns that the use of metalaxyl-M and other

systematic fungicides may mask the symptoms of infection and make inspections invalid for

the detection of P. ramorum (Chastagner et a l, 2010; Shishkôff, 2005).

1.3.6.6 Natural products

Some natural products have been found to have potential inhibitory effects on the

germination and growth of zoospores of P. ramorum. Rice bam extract and grapefruit extract

have both been shown to inhibit the sporulation and growth of P. ramorum (Orlikowski,

2004; Widmer, 2008). A previous research demonstrated that the active compounds of

yellow cedar, hinokitol and thymoquinone have an inhibitory effect on P. ramorum zoospores

and potential to be used as spray on foliar hosts (Manter et a l, 2007). Another study carried

out by Nagel and colleagues (2011) showed resistant coast live oaks contained higher levels

of ellagic acid and tyrosol and total soluble phenolic compounds, all o f which have an

inhibitory effect in vitro on four species o f Phytophthora (Nagle et a l, 2011). Another study

by which investigated the chemical defence response of infected oaks showed there was an

association between higher concentrations of phenolic compounds and resistant species of

oak toward P. ramorum (Ockels et al., 2007). Manter et al, (2007) investigated antimicrobial

activity of heartwood extracts of seven western conifer trees against P. ramorum. The results

of this study showed incense cedar, western redcedar, yellow cedar, western juniper and Port-

Orford-cedar exhibited antimicrobial activity. From 13 compounds isolated from the

bioactive extracts, 6 compounds showed antimicrobial activity in bioassay tests. In a field

trial, redcedar woodchips which were placed under infected California bay laurel on the

forest floor for 4 months reduced the accumulation of P. ramorum DNA in the litter

compared with redwood woodchips that its extract showed no activity against P. ramorum.

The authors had previously suggested that antimicrobial woodchips o f yellow cedar can be

36

spread over trails or footpath to reduce the movement and redistribution of the spores from

these areas (Manter et al., 2006).

1.4 Summary and research objectives

A preliminary study on the effect of heat-treatment of pine wood shavings on the survival of

Salmonella showed promising results. The overarching aim of this project therefore was to

examine the extent to which primary heating techniques can enhance the antimicrobial

activity of wood making it more relevant for a particular reason.

The first part of the project focused on pine wood shavings used as animal bedding materials

and the specific objectives were to:

• Develop and optimise the conditions needed to enhance the antimicrobial activity in

pine wood shavings (Chapter 3)

• Evaluate the inhibitory effect on the microbial activity of chicken litter using

ammonia production as a measure o f microbial activity (Chapter 3)

• Undertake chemical analysis and identification o f the bioactive compounds in heated

wood materials (Chapter 4)

• Screen the bioactive fractions for inhibitory effects on Salmonella Enteritidis (Chapter 4)

The second part of the project extended the concept o f inhibitory activity o f heat treated

wood by testing the activity of larch and rhododendron woodchips against the sudden oak

death pathogen Phytophthora ramorum. Specific objectives were to:

• Evaluate the potential of heated woodchips of P. ramorum hosts (larch and

rhododendron) to inhibit P. ramorum growth in the laboratory (Chapter 5)

• Define the bioactive compounds within heat treated pine, larch and rhododendron

• Examine the potential for the application o f the heated materials in the field aS part of

a management strategy to control P. ramorum

37

CH APTER 2: M ATERIALS & M ETH O DS

2.1 Bacterial isolates and media

Salmonella Enteritidis ATCC 4931 was the model organism used in all experiments when

assessing antimicrobial activity o f heated and non-heated wood materials. Initially, SE was

subcultured on Nutrient Agar (25°C, 48 hours) from the original culture supplied by the

microbiology laboratory. Faculty o f Health and Medical Sciences, University o f Surrey.

After, developing the thermal regime, other microorganisms were also investigated including:

Staphylococcus aureus ATCC 25923, Esherichia coli ATCC 11775, Saccharomyces

cerevisiaeV^CC \9Q%.

2.1.1 Solid agar and liquid media

Fresh cultures of all bacteria strains were routinely cultured on Nutrient Agar for 48-36 hours

at their optimum growth temperature. An overnight liquid culture of the test strains was

routinely prepared in Nutrient Broth for bacteria strains. Malt Extract Agar and Malt extract

were used to subculture Saccharomyces. Retrieved Colony Forming Units (CFU) recovered

from wood were cultured on XLD agar (Xylose Lysine Deoxycholate agar; Oxoid) for

Salmonella, Nutrient Agar for E. coli and Staphylococcus and Malt Extract Agar for

Saccharomyces under aerobic conditions.

2.1.2 Inoculum preparation

Bacteria strains were cultured in Nutrient Broth and incubated for 16 hours at their optimum

growth temperature routinely while shaking at 2 0 0 rpms and were used as starting inoculum

for inoculating wood or filter paper samples (approximately 10’̂ ). For bioassay of the

chemicals, the inoculum size was routinely adjusted at 1 0 ̂ cfu mf^ by measuring the optical

density at 600 nm (ODeoo) using a spectrophotometer.

2.1.3 Bacterial enumeration (cfu ml' )̂

The retrieved numbers of Colony Forming Unites (CFU) from wood samples were

determined by culturing a fixed volume of the suspension (0.1 ml). % strength Ringer’s

solutions was routinely added to one gram of inoculated materials and a 1 0 -fold dilution

series of viable inoculum tested on XLD agar for viability.

2.2 Thermal treatment technique

2.2.1 Wood Materials

The wood materials were provided in the form of 80 cm logs by Forest Research, Famham,

England, and were made into wood shavings by the workshop in Faculty of Health and

38

Medical Sciences, University of Surrey. The materials of different plant species were

individually bagged and kept at room temperature of 25°C for 2 days before they were either

air-dried or heat-treated.

2.2.2 Wood treatment temperature

Pine bark and pine wood were initially ground into a course powder and wrapped in several

layers o f aluminium foil before heating. Wood materials were placed in a muffle furnace

exposed to a range of temperatures (20, 50, 100, 150°C) and heated to the required

temperature for at least one hour. The heated material was allowed to cool before use in

experiments.

2.2.3 Wood treatment time

After the determination o f the efficient temperature, the effect of different heating times o f 1,

24, 48, and 36 and 72 hours at this temperature on the induced inhibitory effect o f heat

treated wood in contact with Salmonella was then assessed.

2.2.4 Wood species

Other wood materials derived from eight different species were also assessed for their

antimicrobial activities after heating. Species tested included: eucalyptus (Eucalyptus

gunnii), beech {Fagus sylvaticd), birch (Betula pendula), ash {Fraxinus excelsior), sweet

chestnut {Castanea sativd), red oak (Quercus rubra), cypress (Cupressus sempervirens) and

spruce (Picea abies).

2.3 CO2 production

For measuring microbial respiration, CO2 evolution within the prepared litter treatments was

determined using an ADC.225.MK3 infra-red gas analyser (Analytical Development Co. Ltd,

Hoddesdon, England). To accurately measure microbial activity, before measuring, all the jar

lids were removed and the head space was air flushed using the air pump on the analyser.

Then to allow sufficient accumulation of CO2 in the jars, the lids were replaced and

tightened. They were left for 60 minutes before the head space of the jars was extracted

using a 60 ml syringe (via a sampling port fitted above the head space). The extracted gas

from each treatment was injected into the gas analyser. A sample of ambient air was used as

a control. Dilutions were made where necessary with N 2 to bring the concentration o f the

samples into line with the measurement range o f the analyser. After the measurement, the

litter treatments were air flushed before incubation. The following equation was used to

39

convert CO2 concentration in the samples to CO2 production per hour per gram of the

materials (h'^ g'^).

CO2 production h'^s'LSamnle concentration (mg I'h - Air concentration (mg I'h

Incubation time (minutes) x Sample weight (g)

2.4 Production of ammonium ions

The concentration of ammonium ions in the litter treatments was measured using an

ammonium probe. One gram of each litter treatment was dissolved into 10 ml o f R.O. water

of a McCartney bottle. Samples were gently swirled around for one minute before the probe

was used to measure the conductivity of the sample. Since, the probe was not selective for

ammonium ions, other ions can interfere with the measurements, notably K^. However, as

the experiment was conducted in the pine wood shavings, the concentration in the

material were too low to cause any significant interference. To measure the actual

ammonium ion concentration in the bedding, a standard curve was made. For this purpose

ammonium chloride was dissolved in R.O. water and a concentration range of 1, 5, 10, 50,

100, 500, 1000, 5000 and 10,000 mg 1"̂ was made. The conductivity of each concentration

was measured and a log linear relationship between conductivity and ammonium ion

concentration was seen. Based on the constructed curve, the actual concentration of

ammonium ions in the litter could be calculated.

2.5 pH

Measurement of pH was carried out by suspending Ig o f each litter treatment in 10 ml of

sterile water. The pH meter (Hanna 210 Instruments, Leighton Buzzard) was calibrated using

pH 7.01 and pH 4.01 standard solutions provided by the manufacture. Between

measurements, the electrode was washed with distilled water. The pH readings were taken

after the mixtures of wood shavings and water had been gently swirled around for one

minute.

2.6 Chemical analysis techniques

2.6.1.1 Wood extraction

Three extracts of heated wood were prepared to determine if the compounds responsible for

inhibitory activity were soluble in hot water (90°C), cold water or methanol.

40

2.6.1.2 Water extraction

Material from each set o f heat-treated and air-dried pine shavings (6 g each) were soaked in

water by adding sterile water to shavings, 2 g in 1 0 ml set up in triplicate, using the following

soaking regimes: 24 hours soaking at room temperature for a cold water treatment and 24

hours soaking at 90°C in a water bath for a hot water treatment. The extracts were then

decanted into sterile vials and kept at 4°C before they were tested.

2.6.3 Methanol extraction

A methanol extract used the same procedure as was followed for water extraction, except

using laboratory grade methanol. Samples were soaked for 24 hours on a roller shaker

(SPERAMAX 5, DENLEY Instrument Ltd.) at room temperature.

2.7 GC-MS analysis of heated and non-heated wood shavings

Gas chromatography mass spectrometry analysis of the methanol crude extracts o f heat-

treated and air-dried pine shavings was carried out using an Agilent Technologies, 7890A,

GC system with an Agilent Technologies 5975 inert XL EI/CIM SD with triple axis detector.

The samples were dissolved in chloroform or methanol (depending on solubility), 2pi o f each

sample was injected and volatised at 250°C for 25 minutes. The column was a HP-5-MS, 30

m X 250pm x 0.25 pm, which was heated initially to 50°C for 3 minutes and then to 250°C at

10°C /minute for 2 minutes, and then the temperature was held for 30 minutes.

2.8 Isolation of active compounds derived from the methanol crude extract

2.8.1 Thin layer chromatography

Thin layer chromatography (TLC) was used to choose a solvent system for column

chromatography which induced a good separation o f the components present in the methanol

crude extract of heated wood shavings of Scots pine. 100% Dichloromethane was chosen to

be the first solvent system, to be followed by a gradient elution using a mixture o f methanol

and dichloromethane. For this purpose methanol was added to dichloromethane step wise in

1 % incremental steps.

2.8.2 Column chromatography

The methanol crude extract of Scots pine (30 g) was dissolved in 100% methanol and mixed

with 10 g silica gel (Merck 9385) followed by a drying step in the fume hood for 2 days. A

50 cm cylindrical gravity column with a 4 cm internal diameter was packed using a slurry o f

silica gel and 100% hexane. The dried crude extract/silica gel was added to the packed

column and eluted using 100% dichloromethane. The eluent was collected in 75ml portions

41

giving around 260 fractions. Thin Layer Chromatography (TLC) was used to analyse the

purity of each fraction. Subsequently, fractions that had similar TLC bands were combined.

A solution of />-anisaldehyde, sulphuric acid and cold methanol (1.5:2.5:96) was used to

visualise the TLC spots. Combined fractions were further purified using small gravity

columns (1cm internal diameter) or preparative TLC plates and each purified product was

identified using NMR spectroscopic techniques. NMR spectra were recorded at room

temperature on either 300 or 500 MHz Bruker AVANCE NMR spectrometer.

2.9 In vitro assays

2.9.1 D irect contact assay

This method was developed to assess the antimicrobial activity of the fractions and

compounds derived from column chromatography. Chemicals were added onto 10 mg of

ground perlite placed inside Eppendorf tubes (1.5 ml). For each fraction, 10 pi was dissolved

in 80:20 ethanol/water and was used to coat the perlite. Four replicates of each fraction were

made. The tubes were left in a laminar flow chamber overnight to allow the solvent to

evaporate. Each tube was inoculated with 1 pi (10^ cfu ml'^) of a log phase culture of

Salmonella Enteritidis. To keep the perlite moist during incubation, 10 pi of sterile distilled

water was dispensed into each tube. As controls, four tubes received distilled water only.

The tubes were incubated overnight and from each treatment tube, a two-fold dilution series

was prepared in Ringer’s solution. For each dilution, 20 pi was plated onto XLD agar to

quantify bacterial survival. This method was used also to evaluate the minimum inhibitory

concentration of purified and identified compounds derived from extracts as well as their

commercial forms.

2.9.2 Synergistic effect assay

To assess the effect o f dehydroabietic acid on the antimicrobial effect o f vanillin, a two-fold

dilution series of 20 mmol stock solution of vanillin purified from fraction Cr 94-100 was

prepared in ethanol and water (80/20: v/v) giving concentrations from 20 mmol to 0.009

mmol. A 20 mmol solution of dehydroabietic acid, which was also purified from the same

fraction, was made in ethanol and water (80/20 v/v). Ten pi of each vanillin dilution was

pipetted onto three, 1 0 mg replicates of ground perlite, thus giving the same concentration per

gram perlite as was in the solution together with 10 pi of 20 mmol dehydroabietic acid. One

set of control treatments did not receive dehydroabietic acid, another only 2 0 mmol

dehydroabieticacid, and a third control treatment received only 1 0 pi ethanol and water at a

ratio of 80/20. Once the alcohol had evaporated, all replicates received 1 pi o f a suspension

42

of Salmonella Enteritidis (10'^ cfu mf^). Tubes were incubated at 25°C for 24 hours before

each particle was dispersed in 1 ml of Ringer’s Solution. From each suspension, six 20 pi

droplets were placed onto XLD agar. The plates were incubated at 25°C for bacterial

enumeration.

2.9.3 pH activity assay

To evaluate the effect o f pH, 20 mmol solution of citric acid (analytical grade, Fisher

Scientific) was prepared in water and the above procedure was followed. The concentration

range for vanillin was kept the same.

2.9.4 Micro Broth Dilution Method

To test the effect of storage pH and temperature on antimicrobial activity of vanillin/DHAA

mixture. Vanillin and DHAA and the mixture stock solutions were made 48 hours before the

start of the experiment. To ensure that DHAA was dissolved two methods of dissolving the

compound were used; in one the compound was dissolved in a phosphate buffer with a pH of

4, in the other treatment DSMO was used with a pH of 7. Two batches of each stock solution

at pH 4 and 7 were made, one was kept in the fridge and the other was kept at 25 °C. The

bioassay test was carried out using a micro-broth dilution technique. Vanillin and vanillin/

DHAA stock solutions were diluted further down in a two-fold dilution series to make a

concentration range between lOmM - 0.625mM. lOOpL o f each concentration was added to

lOOpL of nutrient broth in each well o f a 96 well plate. Three replicates were made for each

concentration. Plates were kept in laminar chamber to allow evaporation o f the solvent..

Subsequently, each well was inoculated with 5 pi of a Salmonella enteritidis suspension

containing 10 ̂ cfu/ml that had grown overnight at 25°C. After inoculation, the plates were

incubated at 25°C for 24 hours. To determine if treatments were bacteriocidal, 0.01 ml from

each well was spotted onto nutrient agar. Plates were incubated for 24-48h and spots with no

bacterial growth were marked as ‘bacteriocidal’. To determine a bacteriostatic effect 50 pi

of lodonitrotetrazolium chloride solution (INT, Fluka, 58030) was added to each well to

determine metabolic activity. After overnight incubation at 25°C no change in colour was

defined as a bacteriostatic effect while metabolism (no effect) was indicated by a red to

purple colour.

43

2.9.5 Bioautographic TLC

The biological activity of the methanol crude extract of pine and active fractions were

evaluated using the direct bioautographic bioassay by Homans and Fuchs (1970) with a slight

modification. TLC plates were cut into square pieces (5cm x5 cm) and aliquots of the test

compounds (20 pi) in ethanol (5 mmol) were spotted in the centre of individual plates

separately using a micropipette. For treatments containing dehydroabietic acid, a mixture of

the two compounds was made (compound 5 mmol, 20 mmol DHAA) and spotted instead.

TLC plates were left overnight inside a fume hood to dry. A 10 ml cell suspension (10'^) of

all test microorganisms were prepared and sprayed evenly over TLC plates inside a fume

hood. Plates were incubated inside a Pyrex tank containing a moist cloth at room temperature

(25°C) for 3 days. Plates were sprayed over with 5ml water solution of iodonitrotetrazolium

chloride dye (INT, Fluka, 58030) each and incubated for 8 hours. Clear zones of inhibition

were measured from the centre o f TLC plates and mean area of inhibition was calculated.

2.10 Determination of the effect of heating of rhododendron and larch wood shaving on

P. ramorum

2.10.1 Wood species of tested materials

P. ramorum infects both larch and rhododendron, therefore, wood chips from rhododendron

{Rhododendron ponticum) and Japanese larch (Larix kaempferi) were heated and their

antimicrobial properties were assessed for development of an inhibitory mulch layer to

control the spread of P. ramorum in areas that were targeted for clearing.

2.10.2 Preparation of crude extracts

To extract the chemical in the heated woods using chromatography techniques, first wood

from Scots pine {Pinus sylvestris), rhododendron {Rhododendron ponticum) and Japanese

larch {Larix kaemopferi) were processed into fine shavings {ca 500 g for each wood type)

wrapped in aluminium foil and placed in a dry oven. Subsequently, the wood shavings were

extracted with methanol by following the procedure in 2.6.3 (Figure 2.1).

2.10.3 Test isolates

Two isolates of P. ramorum originating from two different hosts were selected from the P.

ramorum collection held at Forest Research, Famham, Surrey. Isolates P I578 and BRCOl

had been isolated from rhododendron and Japanese larch respectively. Both isolates belong

to the EUl lineage o f P. ramorum and later, one isolate from the EU2 lineage, P2460, was

also included in some of the in vitro assays.

44

Figure 2.1 The m ethanol extracts o f heat-treated (left) and air-dried (right) wood shavings o f pine.The wood materials w ere soaked in laboratory grade methanol (500 gram in 2.5 litres) on a platform shaker for 48 hours. The extracts were sieved through filter paper tw ice and were left inside a fum e hood for a week to dry.

2.10.3.1 Inoculum preparation

Zoospores

To produce a zoospore suspension, P. ramorum was sub-cultured on fifty carrot agar plates.

(50 g peeled, chopped carrots; 15 g technical agar; 1 litre of distilled water). Plates were kept

under continuous fluorescent light at room temperature for two weeks. Subsequently, 5ml of

distilled water was added to each plate and with a plastic spreader gently swirled around the

plates to dislodge the sporangia from the mycelia. The water containing the sporangia was

pipetted into a sterile beaker and kept in the fridge for 2 0 minutes, then moved to room

temperature for 45 minutes to release the zoospores. Spores from each isolate were counted

four times, using a haemocytometer according to Forest Research Standard Operating

Procedure (Forest-Research, 1997)

Chlamvdospores

Chlamydospores were prepared by the method o f (Tooley et a i. 2008). A spore suspension

of lO' ̂ zoospores m f' in sterile water (300 ml) were prepared and transferred to a zip-lock

plastic bag. Rhododendron leaf disks ( 6 mm diameter) were cut using a cork borer. Two

hundred leaf disks were added to the spore suspension and bags were sealed and kept at room

temperature for 5 days. The water content of each bag was removed using a sieve and the

leaf disks were rinsed with sterile water three times. Leaf disks were transferred to a

moistened tissue towel and placed inside a plastic container. The two containers were

incubated in the dark at 20°C for 4 weeks. Chlamydospores were observed on the leaf disk

using a light microscope.

45

Mycelia as the source of inoculum

A zoospore suspension (concentration of 10"̂ zoospores mf^) was made for each test isolate

and the wells of a 96-well plate were pre-loaded with 100 |il of sterile water per well, 10 pil of

spore suspension then added to each well, and the plates incubated in the dark for 3 days.

The mycelia forming inside the wells were examined after three days under a binocular

microscope and transferred to pre-loaded 96-well plates with known concentrations of the

crude extracts as well as the chemicals derived from the heated wood of the selected species.

2.10.4 In vitro assays

Micro broth dilution method

The inhibitory activity of crude extracts obtained from heated pine, rhododendron and larch

was tested on two isolates of P. ramorum using a micro broth dilution method. For this test,

each well of a 96 well micro titre plate was filled with 0.96 ml carrot broth. A stock solution

of 50 mg ml" ̂ o f each crude extract was made in acetone. A two fold dilution series (25,

12.5, 6.25 mg extract ml'^) was prepared and pipetted into the broth. Three replicates were

prepared for each concentration. Control wells received only acetone and carrot broth. Plates

were left in a laminar flow cabinet for 5 hours to allow the solvent to evaporate. A spore

suspension o f P. ramorum was prepared (as described in 2.10.3.1). Each well was inoculated

with 5 pi of a 10"̂ zoospores ml'^ spore suspension. After inoculation, the plates were

incubated at 20°C for 24 hours. To determine if treatments were fungicidal, 20 pi from each

well was spotted onto carrot agar plates which were incubated for 24-48 hours. Inoculum

spots with no growth were marked as positive fungicidal test results.

2.11 Data analysis

The data were analysed by a one-way Analysis o f Variance ANOVA, GraphPadPrism,

version lnStat3 (GraphPad-Software, 1998). The Tukey Post Analysis Test was performed to

assess the least significance difference between means at a P<0.05 level.

46

CH APTER 3: EVALUATIO N OF H EAT-TREATED W O O D AS LITTER

3.1 Introduction

Poultry litter is known to harbour high numbers of bacteria (Dumas et a l, 2011; Li et a l,

2003; Scheffer, 1965; Terzich et a l, 2000) and the total bacteria concentrations can vary

fromlO^^ and 10̂ ̂ colony forming units per gram of litter (Scheffer, 1965; Terzich et a l,

2000). Among these, pathogenic bacteria causing food-borne diseases are o f great concern to

the public and the poultry industry. Bacteria belonging to the species Salmonella are the

leading cause of intestinal diseases in humans and animals worldwide (Castiglioni Tessari et

a l, 2012). The annual report on zoonoses and food-home outbreaks in the European Union

shows Salmonella accounted for 99,020 reported human cases in 2010 (EPSA, 2010a).

Nearly 26% of Salmonella infections in the UK were associated with consumption of poultry

meat in 2009 (Defra, 2011).

Poultry litter is shown to play an important role in Salmonella survival and transmission in

broiler houses (Doyle and Erickson, 2006; Tucker, 1967). Tucker (1967) found that cycling

of Salmonella between the intestinal tract and litter seems to be significant in maintaining

intestinal infection of broilers. Salmonella can survive for up to 26 months in poultry litter

and spread the contamination among the flock in built-up litter. Higher carcass

contamination is also reported to be correlated with higher contamination o f Salmonella in

the litter during grow-out periods (Volkova et a l , 2009).

Extended survival of Salmonella and the high risk of its introduction into broiler units makes

control difficult. There is no single control strategy which is able to guarantee Salmonella-

free chickens. In the UK, DEFRA welfare codes of practice for prevention and control of

Salmonella are continuously updated to inform farmers and producers about the latest

changes and implementation. In 2011, the disease mitigation and control evidence plan

provided by DEFRA highlighted the need for on-farm cost-effective innovation and the

development of control strategies for zoonotic diseases based on current knowledge in Great

Britain (Defra, 2011). Although, national control programmes o f Salmonella have been

successful in reducing Salmonella incidences, further research is needed to avoid the risk of

new Salmonella strains becoming prevalent and possible changes of pathogen serovors

(Defra, 2011; Doyle and Erickson, 2012).

Ammonia production and volatilization in poultry units is another major health issue for

animals, humans and the environment. The litter condition has a direct influence on the

47

ammonia production level in broiler units. Natural break down of poultry manure by

mineralizing bacteria takes place within the litter environment which leads to ammonia

production and subsequently increasing pH and ammonia volatilization. Application of

acidifying amendments is the most effective current strategy to control pH of the litter

(Marsh, 1999a; Wheeler et a l, 2008). However, use of amendment above manufacturer’s

recommended rate has increased concern about emerging virulent strains of bacteria as a

result o f being exposed to high levels of chemicals (Wheeler et a l, 2008).

Effects of the type of bedding materials on survival of poultry pathogens and ammonia

formation have been studied extensively (Atapattu et a l, 2008; Atencio et a l, 2010; Bilgili et

a l, 1999; Duqueza, 1996; Macklin et a l, 2005; Miles et a l, 2004; Miles et a l, 2011b;

Tasistro et a l, 2007; Torok et o/., 2009). In many cases, the physical parameters o f bedding

materials such as the water holding capacity are found to make a significant difference

between different bedding materials (Atencio et a l, 2010; Bilgili et a l, 1999; Macklin et a l,

2005; Miles et a l, 2011a). Others consider the chemical characteristics o f bedding materials

to be responsible for inhibiting microbial activity (Atapattu et a l, 2008; Duqueza, 1996).

Chopped straw, peanut hulls, rice husks, sand, shredded paper, sawdust, and wood shavings

are commonly used in different countries. Wood materials have been shown to be

particularly efficient in reducing ammonia emissions and improving broiler health and

performance (Miles et a l, 2011b; Torok et a l, 2009). Other research on the survival of

pathogenic bacteria on wood materials have shown that wood exhibits antimicrobial action

against food-borne pathogens such as Salmonella and E. coli (Schonwalder et a l, 2000,

2002). In general, softwoods do not support bacterial growth as well as hardwoods which

may be due to differences in their chemical characteristics (Milling et a l, 2005; Schonwalder

et a l, 2000, 2002). However, choice of bedding materials is limited by what is available on

the market and by season (Embury, 2004). This makes it difficult to implement the

application o f antimicrobial bedding materials and highlights the need for investigating

alternative bedding materials that enhance antimicrobial characteristics.

A review o f the literature has shown that thermal treatments o f wood are well developed to

produce wood with desirable physical and chemical properties. Thermal treatment is mainly

used to improve wood dimensional stability and its resistance to weathering conditions and

decay fungi (Kamdem et a l, 2002; Stamm et a l, 1946; Voda et a l, 2003). Literally several

hundreds of chemicals are produced from wood processing technologies with different

applications in the pharmaceutical and other industries (Mackay et a l, 2009b; Murzin et a l,

48

2007). Up to the present time, research on efficacy or potential applications of heat treated

wood materials and their derived chemicals has been limited to improving the life duration of

wood itself and its resistance toward wood decay fungi.

This chapter aims to describe the development o f a heating regime to enhance the

antimicrobial activity o f pine wood shavings for use as bedding materials in broiler houses.

Due to the significance of food-home diseases caused by Salmonella strains. Salmonella

Enteritidis was chosen as the test organism to evaluate the inhibitory effect o f bedding

materials on Salmonella.

3.2 Experimental

Initially, survival of Salmonella on heated wood materials was tested. In later work to

measure the effect of antimicrobial activity o f treatments on ammonia production, a model

litter was prepared by homogenzing heated wood materials with an aliquot o f litter from a

broiler house to introduce the complex microbial population of chicken manure. Commercial

uric acid was added to provide the microbes with a source of nitrogen.

Full details of the experimental methods and methods o f statistical analysis are given below

and in Chapter 2, sections 2.11.

3.2.1 Wood inoculation

From heat-treated and air-dried materials three batches of one gram wood shavings were

weighed and transferred into McCartney bottles, giving three replicates for each treatment.

Each received 2 ml sterile distilled water one hour prior to inoculation and was then

inoculated with 1 ml of a suspension (10^ cfu ml"^) of Salmonella Enteritidis. Care was taken

to ensure that the suspension was completely absorbed by the test material. Inoculated

shavings were incubated at 25°C for 48 hours. One gram of materials from each treatment

was weighed into a clean McCartney bottle and received 10 ml sterile distilled water. A 10-

fold dilution series (neat - 1 0 '^) was prepared from the shavings and 0 . 1 ml of each dilution

plated onto XLD agar. Plates were incubated for 36 hours at 25°C for bacterial enumeration.

3.2.2 Litter model preparation

Fifty grams of test materials including heat-treated, air-dried and commercial wood shavings,

perlite were placed in 2.5 litre dispo-jars (four replicates for each treatment) that were fitted

with a sampling port for gas analysis. The materials were soaked to 60% of their water

holding capacity, and amended with 1 % spent chicken litter to provide a large microbial

49

inoculum and 1% uric acid that could be converted to ammonia by microbes. All model

treatments were incubated at 25°C for 48 hours to make a homogenized chicken litter (Figure

3.1). Each treatment was replicated four times and samples of gas and material were taken

every three days to assess microbial respiration (CO? evolution), ammonium ion

concentration and the pH of the material. Litter and air samples were taken 4, 7 and 11 days

after incubation and tested for ammonia production and changes in pH.

Figure 3.1 Litter models made in 2.5 liter plastic containersThe containers were fitted with a sam pling port for gas analysis and were kept inside an incubator at 25°C throughout the experim ent.

3.3 Results

3.3.1 Effect of heating regime on the antim icrobial properties of pine bark

The effect o f temperature on the antimicrobial properties o f pine shavings was assessed by

heating ground pine bark and pine wood shavings for 24 hours at temperatures ranging from

50 to 200°C. Antimicrobial activity was assessed by estimating the survival o f Salmonella

Enteritidis in the treated material 48 hours after inoculation. The survival of bacteria in the

heated bark decreased by 2 log units when the temperature o f the treatment increased from

50°C to 100°C (P<0.05) and was completely inhibited on pine wood shavings which had been

heated to 150°C and 200°C in contrast to the outcome at 50°C (P<0.0()1) (Table 3.1).

Table 3.1 Effect o f different heating tem peratures on the recovery o f SE from ground pine bark

Tem perature "C (n=3) Recovery o f SE (log cfu g ‘ ± SD)50 6.30 ± 0.04"*100 4.29 -t 0 .04”150 0.00 1: 0.00"200 0.00 t 0 .00“Significance PO.OOl

^D ifferent letters signify significant differences (P<0.05) between means. (D etection limit= 2 log cfu g ‘)

50

However, when survival of Salmonella on air-dried wood shavings was compared to that on

filter paper it was reduced by one log unit (Table 3.2). These data indicate that heating either

bark or wood shavings o f Pinus sylvestris significantly enhanced antimicrobial activity, but

only when the wood products were heated to a temperature above 100°C.

Table 3.2 Effect o f source materials on the number o f Salm onella Enteritidis recovered from pine shavings before and after heat treatment. (Detection limit= 2 log cfu g'^)

Source m aterials (n=3) R ecovery o f SE (log cfu g' ̂± SD)Air-dried pine shavings 5.3 ± 0.05"*150 °C 0.00 ± 0 .0 0 ”200 °C 0.00 ± 0.00”Filter paper 6.45 ± 0.07“Significance P <0.001

*Different letters signify significant differences between means (P <0.05).

For the control, treatments consisting of shredded filter paper were inoculated and Salmonella

recovery was measured by enumeration o f colony forming units per gram of shredded filter

paper. Salmonella survival on the heat treated filter paper decreased significantly as

temperature increased (P<0.001). When filter paper was heated to 100, 150 and 200°C, log

reduction of 0.4, 2.5 and 2.37 log units were achieved compared to that at 50°C. When filter

paper and pine shaving treatments were compared after treatment at 150°C or 200°C, the

survival of Salmonella was considerably at higher levels on filter paper (4 log units) to that

on heated wood shavings (zero survival). Overall, some Salmonella survived on filter paper

regardless of the temperature applied (Table 3.3).

T able 3.3 Effect o f different heating temperature on the number o f Salm onella Enteritidis recovered from filter paper.

Tem perature “C (n=3) R ecovery o f SE (log cfu g"̂ ± SD)50 6.88 ±0.05"*100 6.48 ± 0.05”150 4 .5 1± 0 .06”200 4.38 ± 0.03“Significance P <0.001

*Different letters signify significant differences between means (P <0.05)

3.3.2. Effect of heated pine shavings on microbial breakdown in modelled litter

Pine shavings were treated at temperatures of 150°C and 200°C for an hour. Wood shavings

were mixed with 1 % uric acid (to provide the nitrogen source) and 1 % of chicken bedding (to

provide inoculum) and were then incubated for 48 hours to create a homogenized litter. The

pH o f the litter material was measured to determine the inhibitory effect o f heat treatment on

51

the microbial breakdown of uric acid in the litter. Perlite and air-dried pine shavings were

modelled as control litters for eomparison.

Table 3.4 Effect o f heating at 150°C and 200°C on the pH o f pine shavings made into litter.

M odelled litter (n=4)

pH

4 days 7 days 11 daysPerlite 6.43 ± 0.05"* 8.33 ±0.02" 9.48 ±0.01"

A ir-dried pine shavings 6.68 ±0.03" 6.67 ±0.08” 8.75 ±0.10"”1 5 0 T 4.29 ± 0.03” 4.60 ± 0 .2 3 “ 8.27 ± 0 .4 1 ”2 0 0 T 5.84 ± 0 .1 f 7.27 ± 0.42"” 9.13 ±0 .02" ”

Significance P<0.001 P <0.001 P<0.001

^D ifferent letters signify significant differences between m eans (P <0.05)

As shown in Table 3.4, after 4 days the pH of the eontrol litters (perlite and air-dried

shavings) was signifieantly higher than the pH of both litters made with heat-treated shavings

heated to 150°C and 200°C (P<0.001 ). After 7 days, the pH value o f the heated pine shavings

at 200°C increased to 7.2 whieh was not significantly different from the pH of perlite and air-

dried shavings. In contrast, the litter with pine shavings heated to 150°C had the lowest pH

value of 4.6 (P<0.001 ). After 11 days, there were no significant differences between the pH

of air-dried or heat-treated litters at either 150°C and 200°C. Overall eomparison, wood

shavings heated to 150°C maintained the lowest pH values after 4. 7 and 11 days of

incubation (Figure 3.2).

pH of m odel litter

109.5

98.5

7.5

^6.5b

5.5 5

4.5 4

3.5 3

♦ perlite

■ air-dried

▲ 150°C

X 200°C

4 6 8 10

Incubation tim e (days)

12 14

Figure 3.2 Change o f pH in m odelled litter over time.

3.3.3 Effect of heated pine wood on CO 2 production from wood shavings based litter (as

indicator of microbial activity)

Heat treated shavings were made into a model chicken litter system (see 3.2.2) to assess the

effect o f heating pine shavings for an hour at either 15()°C or 200°C on microbial activity.

52

Controls consisted of perlite and air-dried wood shavings treated as described above.

Microbial activity was assessed by measuring CO2 production as an indicator of microbial

activity (mg CO iper kg of litter per hour) after 4 ,1 and 11 days.

As shown in Table 3.5, after 4 days o f incubation at 25°C, both 150°C and 200°C heated pine1 1supported significantly lower bacterial respiration rates (6.3 and 103 mg CO2 kg' h' )

compared with the two controls, air-dried pine wood (146 mg CO2 kg'^h'^) and perlite based

litter (272 CO2 mg kg" ̂ h'^) (P<0.001). After 7 and 11 days, the microbial respiration of

150°C heated pine was also significantly lower than perlite, air-dried and heat treated pine at

200°C (P<0.001). Although heated pine at 200°C had a higher microbial respiration rate

compared to 150°C, after 7 and 11 days it supported significantly lower microbial respiration

(174; 195 mg CO2 kg'^ h'^) compared to air-dried (241; 260 mg CO2 kg'^ h'^) and perlite ( 359

mg CO2 kg'^ h'^) treatments.

Table 3.5 Effect o f heating at 150°C and 200°C for one hour on the microbial activity expressed as CO2

production in a pine shavings base litter.

M odelled litter (n=4)

R espiration (m g CO 2 kg^ h'^)

4 days 7 days 11 daysPerlite 272 ±26"* 359.7± 20" 128.6 ± 8.2"Air-dried Pine shavings 146 ±10” 241 ± 13.4” 260.4 ± 1 1 .7 ”150°C 6.3 ± 3.2“ 32.1 ± 4 .5 “ 89.1 ± 5 .3 “200°C 1 0 3 ± 1 8 ” 174 ± 2 5 ” 195.8 ± 2 3 .2 ”Significance P<0.0 01 P<0.001 P<0.001

*Different letters signify significant differences between means (P <0.05)

3.3.4 Effect of heating duration at 120®C on CO2 production, pH and ammonia

concentration

The effect of heating pine shavings at 120°C for increasing periods o f time (1 to 72 hours) on

the antimicrobial activity of the treated shavings was measured, using the model chicken litter

system (see 3.2.2). Microbial activity was assessed by measuring microbial respiration (CO2

evolution), rise in pH (as a result of ammonium ion formation) and ammonium ion

concentrations in the litter after 4, 7 and 11 days incubation at 25°C. The results are

presented in Tables 3.6, 3.7 and 3.8.

53

Table 3.6 Effect o f heating pine shavings at 120°C for increasing periods o f time (1 to 72 hours) onantimicrobial activity expressed as CO2 production.

Base m aterials o f M odelled litter (n=4)

R espiration (m g C O 2 kg'^ h'^)

4 days 7 days 11 daysPerlite 20.6 ± 4.3 12.9 ± 0 .5 6 13.5 ± 1.9Commercial shavings 85.5 ± 2.4 107.9 ± 18.8 67.5 ± 4.5Air dried shavings 501.2 ± 105.4 143.7± 9.4 115.4 ± 2 .71 hour heating 52.0 ± 47.5 134.2 ± 3 4 .4 117.3 ± 11.63 hours heating 11.3 ± 6 .2 107.5 ± 25.6 105.0 ± 16.66 hours heating 1.2 ± 1.2 157.6 ± 7 9 .1 118.8 ± 5 .724 hours heating 4.3 ± 1.8 211.1 ± 2 0 .5 102.0 ± 11.472 hours heating 0.0 ± 0.0 3.4 ± 1.8 76.0 ± 6.3Significance P<0.001 P<0.001 P<0.001

Table 3.6 illustrates that the total microbial respiration decreased as pine shavings were

heated for longer periods of time. After 4 days incubation CO2 production in pine shavings

that had been heated for 3 and 6 hours was lower (11.3; 1 . 2 mg CO2 kg'^ h'^) than the air-

dried and commercial shavings (501.2; 85.5 mg CO2 kg'^h'^). For pine shavings which had

been heated for 24 and 72 hours at 120°C significantly less (P<0.001) CO2 was produced (4.3

mg CO2 kg'^ h'^ and 0 . 0 0 mg CO2 kg'^h"^) compared to the two controls, perlite and

commercial shavings, which produced 2 0 . 6 mg CO2 kg" ̂ h'^ and 85.5 mg CO2 kg'^ h'^

respectively. Heat treatment of 72 hours resulted in the lowest CO2 production compared to

the 24 hours treatment particularly after 4 and 7 days incubation, but also to a lesser extent

after 1 1 days of incubation (76 vs. 1 0 2 mg CO2 kg'^ h" )̂ (P<0 .0 0 1 ).

Table 3.7 Effect o f heating pine shavings at 120°C for increasing periods o f time (1 to 72 hours) on antimicrobial activity o f the treated shavings expressed as the pH.

Base m aterials o f M odelled litter (n=4)

pH values

4 days 7 days 11 daysPerlite 8.31 ± 0 .0 5 9.11 ± 0 .0 6 8.72 ± 0 .0 7Commercial shavings 6.14 ± 0 .0 1 6.32 ± 0 .0 9 8.53 ± 0.05Air dried shavings 6.15 ± 0 .0 1 7.81 ± 0 .2 8 8.51 ± 0 ,1 21 hour heating 5.93 ± 0.02 5.33 ± 0.35 8.03 ± 0.443 hours heating 5.79 ± 0 .0 2 5.20 ± 0.05 7.47 ± 0.306 hours heating 5.97 ± 0.06 4.65 ± 0.22 7.17 ± 0 .3 024 hours heating 5.15 ± 0 .0 9 4.55 ± 0 .1 2 6.65 ± 0 .2 572 hours heating 5.01 ± 0 .0 4 4.13 ± 0 .0 8 4.75 ± 0 .3 1Significance P<0.001 P<0.001 P<0.001

Table 3.7 and Figure 3.3 indicate the effect of different exposure times of pine wood

shavings to a temperature of 120°C on the pH of the model litter over time. Heated shavings

were made into litter and incubated 4 days before the first measurement.

54

After 4 days the pH of litter treatments made with shavings and heated from I to 72 hours

was lower compared to litter treatments made with perlite or air-dried and commercial

shavings. Litter made with pine shavings which was heat treated for 24 or 72 hours had the

lowest pH at 5.15 and 5.0 compared to perlite which had the highest pH of 8.3. Commercial

and air-dried shavings had the same pH of 6 .1. After 7 days, the pH had decreased in all litter

treatments made with heated pine, while the pH of air-dried, commercial and perlite litter

increased. Between 7 days and 11 days of incubation there were no significant differences

between the pH o f commercial, air-dried and treatments with 1-24 hours o f heating. In

contrast, the 72 hour treatment had a significantly lower pH of 4.7 (P<0.001). Air-dried

shavings had an alkaline pH even after 7 days incubation. In general, the increase in pH over

time was inversely related to heating time with acidic conditions being maintained for longer

as heating time increased (Table 3.7).

I pH of m odel litter10 1

9.5 - 9

8.5

7.5 7

a 6.5 6

5.5 5

4.5

4 13.5

3 4

ftX

$+

♦ perlite

■ commercial shavings

A air-dried shavings

X 1 hour heating

X 3 hours heating

• 6 hours heating

+ 24 hours heating

- 7 2 hours heating0 12

incubation tim e (days)

Figure 3.3 Com parison o f pH between 8 different model litters over tim e (n=4).

As shown in Table 3.8. the amount o f ammonium ions in pine shavings decreased when the

shavings had been pre-heated for increased periods o f time. In comparison to commercial

and dried wood shavings, the ammonium ion production rate is considerably lower when

shavings were heated for 24 or 72 hours.

3 0

Table 3.8 Effect o f heating pine shavings at 120°C for increasing periods o f time (1 to 72 hours) onantimicrobial activity o f the treated shavings expressed as ammonium ion concentration.

Base m aterials o f M odelled litter (n=4)

A m m onium ion concentration (m g kg'^)

4 days 7 days 11 daysPerlite 1058 ± 7 5 455 ± 24 303 ± 17Commercial shavings 50 ± 0 508 ± 1 9 590 ± 42Dried shavings 8 0 ± 14 360 ± 27 3 1 0 ± 91 hour heating 8 0 ± 11 133 ± 96 315 ± 5 43 hours heating 70 ± 7 2 0 5 ± 152 378 ± 756 hours heating 1 1 7 ± 5 43 ± 5 323 ± 3324 hours heating 132 ± 4 0 75 ± 2 0 275 ± 1972 hours heating 90 ± 6 7 3 ± 10 108 ± 49Significance P<0.001 P<0.001 P<0.001

Wood that was heated for 72 hours at 120°C had the lowest concentration of ammonium ions

after 4, 7 and 11 days of incubation (90, 73, 108 mg ammonium ion kg’ ̂ respectively)

compared with perlite after 4, 7 and 11 days (1058, 455, 303 mg ammonium ion kg'^

respectively) and wood shavings that had been heated for 24 hours after 4, 7 and 11 days

(132, 75, 275 mg ammonium ion kg'^ respectively). These differences were highly

significant (P<0.001) (Table 3.8).

3.3.5 Spectrum of enhanced antimicrobial activity of heated pine shavings

The antimicrobial activity of heat treated pine shavings (120°C for 72 hours) was tested

against two other pathogens that are frequently isolated from poultry litter. Staphylococcus

aureus and Escherichia coli. The inhibitory effect o f heated shavings on each pathogen was

determined by inoculating one gram of heated shavings with 1 0 ̂ cells o f the test strains.

Numbers of the bacteria recovered from heated shavings was determined by enumeration of

colony forming units on agar plates. The results are shown in Table 3.9. Survival of both

pathogens was completely inhibited on heated shavings as compared with air-dried shavings

and filter paper (P< 0.001). Survival of both pathogens was higher on air-dried shavings

compared to that on filter paper.

T able 3.9 Survivals o f S. aureus, and E. coli and on heat treated and air-dried wood o f pine and air dried and heated filter paper. (Detection limit= 2 log cfii g'^)

Survival (log cfu g' ̂± SE)

Tested strains (n=3) H eated pine A ir-dried pine Filter paper SigniHcance

Staphylococcus aureus ATCC 25923 00 ± 0 0 7.78 ± 00 5.0 ± 00 P<0.001

Escherichia coli ATCC 11775 00 ± 0 0 7.34 ± 0 .0 2 6.72 ± 0 .0 1 P<0.001

56

3.3.6 Evaluation of the antimicrobial activity of different wood types after heating

A heating regime of 120°C for 72 hours was applied to wood shavings from eight different

tree species as follows: pine, spruee, cypress, ash, beech, red oak, bireh, eucalyptus. The

extent of antimicrobial activity was determined by inoculating 1 gram of each wood type with

10 ̂cells of Salmonella Enteritidis. Survival was measured after 48 hours incubation at 25°C

(Table 3.10). Heating at 120°C significantly inhibited the growth o f Salmonella on wood

shavings of pine, cypress, ash, beech, red oak and bireh (zero recovery) compared to survival

on air-dried wood shavings made from the same tree species (P<0.001). There were also

small, but significant differences in the antimicrobial properties of air-dried wood shavings.

Spruce, birch and oak appeared to have the least antimierobial activity, with large numbers of

bacteria (>10^ colonies g'^) recovered from the wood shavings of these tree species. Cyprus

and eucalyptus appeared to be the most antimicrobial before heat-treatment, with almost two

times less recovery of Salmonella compared with spruce. Heating resulted in a significant

increase (P<0.001) in the antimicrobial activity of all of the wood species that were tested,

resulting in a 4 fold increase in the antimierobial activity of eucalyptus wood shavings, a

1000 fold increase in antimicrobial activity in spruce, and complete inhibition of Salmonella

with pine, Cyprus, ash, beach, oak and birch (Table 3.10).

Table 3.10 Recovery o f Salm onella Enteritidis from inoculated heated and air-dried wood shavings derived from 8 different wood species.

W ood type (n=3) A ir-dried (log cfu g'b H eated (log cfu g'b SignificancePine (soft) 6.88 ± 0 .1 1 “* 0

Spruce (soft) 7.56 ± 0.06” 4.50 ± 0 .0 2 P<0.001Cypress (soft) 5.24 ± 0.07” 0

Ash (hard) 7.12 ± 0.06“ 0Beech (hard) 6.85 ± 0 .1 9 “ 0

Red Oak (hard) 7.22 ± 0 .1 3 “ 0Birch (hard) 7.32 ± 0.04“*̂ 0Eucalyptus 5.93 ± 0.07” 5.3 ± 0.06 P<0.001

Significance P<0.001 P<0.001

*Different letters indicate a significant difference (P<0.05) between means.

3.3.7 Effect of time of exposure to heat treatment of 120”C on pH of pine wood shavings

Wood shavings of pine were heated for 0, 0.5, 1, 2, 3, 4, 5, 6 , 7, 8 , 24, 30 and 48 hours and

the pH of each treatment was measured by suspending 2 grams of wood shavings in 20 ml

sterile water before pH was measured. The pH of wood shavings were found steadily to

decrease from an average of 4.6 before heating to 4.2 after 24 and 30 hours of exposure to

120°C. After 48 hours the pH value decreased to an average o f 3. Figure 3.4 shows the pH

57

of pine shavings decreased significantly after 48 hours of heating compared to that before

heating (P<0.001 ).

pH change of heated pine shavings

R2=0.7SE=0.1Cl=95%

♦ pH

16 24

Tim e (hour)

32 40 48

Figure 3.4 Changing pH values o f pine shavings after different tim es o f heat treatm ent.The pHs o f pine wood shavings were measured after 0.0, 0.5, 1, 2. 3, 4. 5, 6, 7, 8. 24, 30 and 48 hours. Linear regression analysis was perform ed in Excel 2010 (95% Confidence Interval).

3.4 Discussion

The first part o f this study aimed at developing a heating technique to enhance the

antimicrobial activity in pine bark and wood shavings that could have application for poultry

bedding materials. The laboratory study showed that heating at different temperatures

positively increased the inhibitory activity o f both bark and pine wood shavings which

reduced Salmonella levels in the shavings. Thus enhanced inhibitory activity increased as

treatment in temperatures raised from 50°C to 200°C (Table 3.1, 3.2). Interestingly, heating

to temperatures above 100°C, completely inhibited recovery o f Salmonella compared to air-

dried shavings (P<0.001). The results of this chapter also demonstrated that untreated pine

wood shavings supported Salmonella survival. In a previous study by Dawson et al. (2007)

on survival and cross transfer o f Salmonella between the environment and food. Salmonella

Typhimurium was able to survive on wood and contaminate food. Warren el al. (2005)

investigated survival o f a cocktail of 5 serovars of Salmonella in different packaging

materials (PVC, stainless steel, sponge roller, conveyor belts and unfinished oak wood) and

found that wood supported the long-term survival o f Salmonella. Based on the literature and

findings of this study, it can be concluded that untreated wood materials support the survival

and transmission o f Salmonella in the environment. Several research studies also have

58

demonstrated a high incidence of Salmonella in new bedding material (Dougherty, 1976;

Long et a l, 1980). Therefore, a pre-heating treatment o f bedding materials may reduce the

risk o f Salmonella contamination in broiler at the time o f placement. This is very important

since younger chickens are more susceptible to Salmonella when placed on new bedding

material and use of contaminated wood shavings may increase the risk of Salmonella

outbreaks on-farm.

Further experiments on the effect of exposure time to heat treatment showed perlite and dried

shavings supported significantly higher levels of respiration compared with shavings given an

hour of heat treatment. However, dried shavings and 1 hour heat-treated shavings did not

differ in the amount of ammonia they produced (Table 3.8). The effect o f time exposure on

enhanced antimicrobial activity showed that longer time exposures to a temperature of 120°C

significantly decreased microbial respiration of the model litters when compared to perlite,

air-dried and commercial shavings litter over an 11 days test (Table 3.6). The differences

were more distinctive between the exposure times after 4 days, with 3, 6 and 24 hours

showing the most variation between 4 and 7 days o f incubation. However, the differences

between all heat treated shavings were not significant except with the 72 hour o f heat

treatment sample (Table 3.6). The 72 hours heat treatment sample also had the lowest

microbial respiration rate after 11 days o f incubation. Although, the inhibitory activity of

heated wood o f pine on mineralizing bacteria was not directly evaluated, the lower respiration

rate from heated wood material indicates that the activity o f microorganisms in the litter was

inhibited, including mineralizing bacteria. In addition, ammonia formation in heat treated

wood shavings samples was also significantly different from air-dried, commercial shavings

and perlite litters after 7 and 11 days, and particularly low for 24 and 72 hours exposure

(Table 3.8). Ammonia production of commercial shavings and air-dried shavings was

approximately 9-10 times and 5 times more than o f the 6 , 24 and 72 hours heat treated

shavings after 7 days (Table 3.8). Variation in microbial respiration in the 3, 6 and 24 hours

treatment was also reflected in ammonia production. However, after 11 days only the 72

hour heat treated shavings had a significantly lower value (108 mg ammonium ion kg'^)

compared to that of commercial shavings with a value of 590 mg ammonium ion kg (Table

3.8). In the tested litters, pH was directly correlated with higher ammonia production. These

findings are in agreement with a study by Pop and Cherry (2000) which found pH and

ammonia formation were positively correlated. Unexpectedly, pH values of all heat treated

shavings decreased at 7 days of incubation, while the pH of air-dried and commercial

59

shavings increased. In this experiment, 72 hour exposure had the lowest pH and maintained a

lower pH compare to air-dried and commercial shavings over time. In comparison, perlite

litter produced extremely significant levels of ammonia after just 4 days. This can be

explained by fast microbial breakdown over a short period o f time, but the microbial

respiration rates and pH of the perlite litter did not reflect the extreme predicted values.

Although heating pine wood shavings to 200°C was as effective as heating to 150°C in

reducing Salmonella survival, in further experiments when the 200°C treated shavings were

challenged with a complex inoculum of spent litter and uric acid, it resulted in higher

microbial respiration compared with the I50°C treatments (Table 3.5). Also, the model litter

system made with 150°C heated wood maintained an acidic pH after 7 days while the pH

value of that at 200°C reached pH>7 which was associated with a higher microbial respiration

(Table 3.4). It was interesting to note that the pH of perlite was not significantly different

from the pH of the air-dried shavings, although microbial respiration of perlite was

significantly higher. This may be related to a lower microbial activity on air-dried shavings

compared to perlite, but as the pH values are the same it may also indicate that the activity of

the mineralizing bacteria is unaffected by substrate.

Overall, a heating regime of I20°C for 72 hours was found to be optimum for enhancing the

antimicrobial effect in pine wood shavings to inhibit Salmonella, as well as reducing

ammonia formation and the pH of the model litter. The heating technique was also effective

at significantly inhibiting Staphylococcus aureus and Escherichia coli, two other pathogens

known to be present in poultry litter (Table 3.9). In addition. Salmonella hsid lower survival

on filter paper compared with air-dried shavings, which may be due to a lack o f nutrition in

the filter paper substrate (Table 3.2).

The heating technique also significantly increased antimicrobial activity o f hardwood

shavings o f ash, beech, red oak as well as softwood species such as pine and cypress

(P<0.001) (Table 3.10). This highlights the potential of heat treatment to enhance the

antimicrobial properties of hardwoods and use these as substitutes when there is a shortage o f

the most desirable wood materials as bedding for poultry. Air-dried eucalyptus and cypress

had also an inhibitory effect on Salmonella recovery compared to the other tested wood

species.

It is also noteworthy that the pH of the wood shavings after 24 hours o f heat treatment was

found to be lowered from an average of 4.8 to 4.2. Although, pH values o f less than 5 are

6 0

known to be unfavourable for Salmonella, its survival in treated litter with extremely acidic

pH as low as 2.1 has been recorded (Williams, 2012). Thus, it seems unlikely that only lower

pH of heat treated wood completely inhibited Salmonella growth. However, low pH in

combination with low water activity can be effective at reducing Salmonella survival in the

litter. In a previous study, Payne et al. (2007) achieved a 5 log unit reduction o f Salmonella

at pH 4, although this was combined with maintaining water activity at less than 0.84. To

maintain a water activity o f 0.84, a moisture content of 25-30 % is required (Ye et a l, 2006).

In the present study, moisture content o f the various wood treatments was set at 60% of their

water holding capacity, which is equivalent to a water activity of 1 (Ye et a l, 2006),

indicating the inhibitory activity of heated wood materials against Salmonella is in addition to

the physical parameters. However, the lower pH o f heated wood can be considered as an

advantage over air-dried materials, as it reduces the need for application o f litter amendments

in controlling pH.

In the next chapter chemical characteristics of heated wood of pine which were effective at

inhibiting Salmonella and microbial activity in the modelled litter, by reducing ammonia and

pH, will be investigated to determine the bioactive compounds responsible for enhanced

antimicrobial activity.

61

CHAPTER 4: CHEMICAL ANALYSIS AND EVALUATION OF ENHANCED ANTIMICROBIAL

ACTIVITY OF HEAT-TREATED WOOD

4.1 Introduction

Antimicrobial compounds are essential tools to control microorganisms in the environment so

that the quality o f human life can be maintained and animal health conserved. Industrial

processes, pharmaceuticals, plant health services, animal health services, hospitals, clinics,

water treatment facilities and public access facilities benefit enormously by using a variety of

antimicrobial agents (American-Chemistry-Council, 2010). Antimicrobials can have a

variety o f applications. They are used as medicines to cure or prevent human diseases, to

inhibit the spread of disease through the use of disinfectants and to protect manufactured

products and industrial equipment and systems from deterioration (American-Chemistry-

Council, 2010). They are also used in food and beverages in the form of food additives and

preservatives, to protect and treat natural resources, water supplies and forests through their

used as pesticides. Antimicrobials are of great importance for treatments o f infectious

diseases ever since they have been discovered. However, in the last decade antimicrobial

resistance has become a serious public health concern because o f the social and economic

implications. Increased mortality rates particularly in infants and the elderly, increase the

cost of treatments and prolong hospital stays and so are major impacts. (Levy, 2001; Martin

et a l, 2010; Shea, 2003)

Overuse of antimicrobials has resulted in the development of resistance in a number of

environments including clinical facilities, farms and hospitals (Martin et a l, 2010). Using

antimicrobials as growth promoters or for therapeutic reasons in poultry, animal husbandry

and agriculture is also known to contribute towards antimicrobial resistance in humans. The

residues o f antibiotics in meat and animal products may result in humans being exposed to

antimicrobials unnecessarily and this may result in resistance in the natural flora in humans

(Kapil, 2005). The use of antimicrobials shifts natural selection towards resistant bacteria in

the intestines of animals intended for consumption. Resistant non-pathogenic bacteria on-

farm can also act as a pool of transferrable genes (DeFrancesco et a l, 2004; Kelley et a l,

1998). Resistant strains of pathogens causing food-borne diseases such as Salmonella are

increasing (Angulo et a l, 2004). Through the increased incidence of resistant pathogens and

the rapid development of the processing industry, there is now an urgent need for new

sources of antimicrobial compounds.

6 2

Many of the well-known antimicrobials were originally derived from plants, bacteria and

fungi. A large numbers o f bioactive chemicals derived from different plant species have been

identified and used to control microorganisms (Davidson et a l, 2005b). Plant materials are

the most important source of natural antimicrobials due to their chemical diversity and mass

availability (Cowan, 1999). Many research studies focus on exploring the potential of new

species o f plants whilst, others review the general techniques and procedures used to discover

novel bioactive substances and their evaluations (MacKay et a l, 2009a). Fermentation,

extraction and torrefacation of wood materials are examples o f developed techniques applied

to produce different chemicals in large quantities, such as solvents and resins. Valuable

chemicals derived from wood by various processes including heating also provide great

advantages to human life, animal health and economic welfare (MacKay et a l, 2009a;

Murzin et a l, 2007). Many of these chemicals are manufactured and used as antimicrobial

agents, food preservatives or intermediate chemicals in pharmaceuticals (Murzin et a l,

2007). Because they are produced as a result o f chemical modification o f wood structural

components, desirable chemicals are produced under the same heating regime regardless of

the chemical differences between wood species. These chemicals are then extracted using

organic solvents and identified using analytical techniques including Nuclear Magnetic

Resonance spectroscopy (NMR) and gas chromatography-mass spectrometry (GC-MS) for

manufacturing.

Although, enormous numbers o f chemicals are derived from wood processing techniques,

research and development around the heating techniques has been focused on improving the

life duration of wood and its resistance toward wood decay fungi (Kamdem et a l, 2002;

Savluchinske-Feio et a l, 2006; Voda et a l, 2003). Therefore, most o f the existing

developments are based on need in the market for commercially profitable products and their

application in sectors such as furniture manufacture and construction. Little has been done in

examining or developing techniques that introduce valuable levels of antimicrobial

compounds which have the potential to inhibit food-borne pathogens. However, the heating

technique developed and applied to pine wood shavings described in Chapter 3, showed

promising results in inhibiting SE when in direct contact. The aim of this chapter was

therefore to investigate chemical properties of heated wood using chemical analysis

techniques.

63

4.2 Experimental

The data presented in Chapter 3 revealed that heat-treated wood shavings of pine exhibited

strong antimicrobial activity compared with air-dried shavings. In order to be able to isolate

and identify the chemicals responsible for the activity, pine wood shavings were heat treated

with the thermal regime developed in Chapter 3 and extracted with cold water, hot water and

methanol. The crude extracts were impregnated into shredded filter paper and inoculated

with SE as described below (4.2.1). After incubation, the survival rate of Salmonella was

measured by the enumeration of colony forming units recovered from one gram filter paper

after incubation at 25°C. The same procedure was applied to air-dried pine shavings for

comparison. To evaluate the change in antimicrobial activity o f heated wood after extraction,

the three types o f extracted wood shavings (cold water, hot water and methanol) were

allowed to dry for 3 days and then inoculated with Salmonella as described below (4.2.1).

After incubation, one gram of each type of extracted wood shavings was tested for the survival

of Salmonella (cfu g'^).

4.2.1 Extract assays

Water and methanol crude extracts of pine wood shaving were prepared as described in 2.5.2

and 2.5.3. For each extract, 1 ml was added onto shredded (autoclaved) filter paper in

McCartney bottles (12 samples, Igram each). Samples were left overnight to absorb the

extracts. Control treatments received 1 ml of Ringer’s solution strength). A suspension of

SB (10 ^cfti ml'^) in Ringer’s Solution was made to inoculate the bottles. Each sample bottle

was inoculated with 1 ml of the suspension and then incubated overnight at 25°C.

Subsequently, a dilution series (10'^ to 10'^) for each sample was plated onto XLD agar to

assess bacterial survival. The plates were incubated at 25 °C and the colony count was used

to compare inhibition against control treatments and extracts from air-dried shavings.

In a second experiment, the antimicrobial activity of heat treated wood shavings after

extraction was tested to prove that extraction removed the chemicals responsible for the

antimicrobial activity. In this experiment the extracted wood samples were left to dry at 50°C

for 3 days. Two grams of each extracted sample was then transferred to a McCartney bottle

and inoculated with a 2 ml suspension of Salmonella. Two grams shredded filter paper was

used as the control. All samples were incubated at 25°C for 24 hours after which time a

dilution series (10'^ to 10'^) was prepared and plated on XLD agar. Plates were incubated

over night at 25°C for bacterial enumeration.

64

4.2.2. Chemical analysis

To be able to test the effect of the dominant chemicals from heat-treated wood individually

against SE, the methanol crude extract was fractionated by gravity column chromatography

and a total of 260 fractions were collected and screened for their activity (2.8.2). Due to the

lipophilic nature of some of the isolated fractions/compounds, a new bioassay technique was

developed to evaluate the bioactivity o f the chemicals using perlite as a base substance

(2.9.1). Chemical analysis of heat-treated pine using GC-MS and NMR spectroscopy was

carried out to identify the chemicals produced (2.7 and 2.8.2). Finally, the spectrum of

potentially active compounds was tested on other pathogenic bacteria commonly found in

animal bedding materials including, Escherichia coli. Staphylococcus aureus and

Saccharomyces cerevisia (Dumas et al., 2011).

4.3 Results

4.3.1 Extract assays of the methanol crude extract

The outcome of testing extracts o f pinewood applied to filter papers inoculated with

Salmonella is shown in Table 4.1. Both cold and hot water extracts of the heated pine

shavings did not significantly affect SE recovery compared to the control. However, the

methanol extract of heated pine shavings significantly reduced the SE numbers compared to

the control and water extracts by approximately 3 log units (P<0.001). Overall, the survival

of SE in all extracts o f air-dried pine wood also showed little significant difference between

the treatments compared with the control.

T able 4.1 Recovery o f SE from inoculated filter paper treated with extracts from heated and air-dried pine shavings.

Treatm ent: Shredded filter paper pre­

coated (n=3)

SE recovery (log cfu g'^) from heated w ood

SE recovery (log cfu g’ )̂ from air-dried w ood

Cold water Extract 7.54 ± 0.05=* 8.14 ± 0.03=’’

Hot water Extract 7.37 ± 0.06= 7.94 ± 0.05*’°

Methanol Extract 4.80 ± O .lfr 7.91 ± 0.1 o'’"

Control 7.63 ± 0.10= 7.72 ± 0.09 "

Significance P < 0.001 P < 0.05

* D ifferent letters indicate a significant difference between treatm ent means

When the Salmonella inoculum was applied directly to extracted pine shavings, a similar

pattern emerged (Table 4.2). No Salmonella was recovered from heated pine shavings which

65

had been extracted with water, either hot or cold. In contrast, there was a significant

difference between the survival of SE on wood shavings which had been extracted with

methanol compared with the control (P <0.001). The SE concentration recovered from

methanol extracted wood was 4 log units higher than the water extracts but 2.6 log units

lower than the control (Table 4.2). For air-dried wood, no significant difference was

observed between water extracted wood and controls but the shavings extracted with

methanol, did yield a slightly but significantly lower number of SE compared with the control

(P<0.05).

Table 4.2 Recovery o f Salm onella (log cfu g'' ± SE) from inoculated, heated and air-dried pine shavings

T reatm ents: (Extracted w ood)

SE recovery (log cfu g’^)/from heated w ood after extraction

SE recovery (log cfu g^)t from air-dried w ood after extraction

Cold water Extract 00.00 =* 7.63 ± 0.12 =

Hot water Extract 00.00 = 8.02 ± 0.05 =

Methanol Extract 4.00 ± 0.00 ’’ 7.06 ± 0.12

Control 7.63 ± 0.10 " 7.72 ± 0.09 =

Significance P < 0.001 P < 0.001

* D ifferent letters indicate a significant difference between treatm ents (Detection Lim it= 2 log cfu g’ )̂)

4.3.2 GCMS analysis of crude extracts of pine

Results of the above experiments indicated that the methanol crude extracts o f heat treated

shavings exhibited antimicrobial properties that significantly reduced the survival rate of

Salmonella. In order to identify which constituents of heat treated wood were responsible for

the enhanced antimicrobial properties, solutions o f the heat treated and air-dried crude

extracts were prepared and subjected to GC-MS analysis. A comparison o f the

chromatograms obtained from the two crude extracts revealed the presence o f peaks on the

chromatogram of the heated crude extract that were missing from the chromatogram obtained

from the non-heated crude extract (Figure 4.1). The NIST library suggested that the new

peaks observed for the heated crude extract at retention times of 14.18, 14.75, 17.03 and

18.80 minutes could be attributed to 4-hydoxybenzaldehyde, vanillin, 4-hydroxy-3-methoxy

benzoic acid and 4-hydroxy-2-methoxy cinnamaldéhyde respectively. The other peaks that

were present in both chromatograms obtained from both the heat treated and air-dried crude

extracts had retention times of 20.98, 22.75, 24.72 and 25.62 minutes and the NIST library

suggested that they were likely to be n-hexadecanoic acid, oleic acid, pimaric acid and

66

dehydroabietic acid respectively. The salient features o f the two chromatograms of the heated

and air-dried crude methanol extracts are marked for comparison in Figure 4.1 (as per the

NIST library).

Piinanc acid

NL:3 20E7 TC ke90-=

aoi

@ so-2473S 4CK

20.96

10 1? 1< 10 18 20 Time (fn in)

10=

nchvdroabieticRT: 0.00 . 30.19

lOOn

90-; vanillin

80=70= iydroxy-3-melhoxy-benzoic acid

z' \ 4-hydroxy-] methoxy-cinamaldeh de

y - 2801ziJdüLih

4-hydroxybenzaldeh\de

D=

Figure 4.1 A com parison between tw o G C-M S chrom atogram s o f heated and air dried pine wood.N ew peaks at retention tim es o f 14.8, 14.75, 17.03 and 18.80 m inutes for the crude extract o f heated w ood that were missing in crude extract o f air dried wood are 4-hydoxybenzaldehyde, vanillin, 4-hydroxy-3-m ethoxy benzoic acid and 4-hydroxy-2-m ethoxy cinnam aldéhyde respectively. Dehydroabietic acid peak at 25.6 m inutes was increased in heated sam ple. In contrast, pimaric acid peak at 22.75 was considerably decreased.

4.3.3 Active fractions from column chrom atography

From a total o f 260 fractions derived from column chromatography, the following (groups)

showed antimicrobial activity against Salmonella Enteritidis and were purified and analysed

further analysed further using GC-MS to identify the compounds responsible for the

antimicrobial activity o f a given group of fractions.

Fraction C r 97-104: TEC analysis identified four compounds in this fraction with anti­

microbial properties. Attempts to purify these compounds resulted in the isolation o f vanillin

(A5 .7 ) and dehydroabietic acid (A 1.3 ) and two other compounds that were inseparable and

67

could therefore not be identified.

Fraction 2Cr 105-109; Purification of this fraction resulted in the isolation o f dehydroabietic

acid, picealactone-A (A4 ) and two other compounds that were inseparable.

Fraction 2Cr 110-115: This fraction yielded dehydroabietic acid, 3, 3’-dimethoxy-4, 4 ’-

dihydroxy stilbene (A 14.16) and also picealactone A (A4 ).

Fraction 2Cr 116-121: This fraction yielded dehydroabietic acid, 3, 3’-dimethoxy-4, 4 ’-

dihydroxy stilbene and acetovanillone (A n.12).

Fraction 2Cr 122-133: This fraction yielded dehydroabietic acid and acetovanillone.

Fraction 2Cr 138-139, Cr 140-160: This fraction yielded dehydroabietic acid and

coniferaldehyde (A 17.19). Dehydroabietic acid (DHAA) and vanillin were found to

prédominante in the methanol extract, but it was not possible to completely purify DHAA

and vanillin.

4.3.4 Yield of different molecules of interest from heated Scots pine wood shavings

As shown in Table 4.3, the most abundant compound isolated from heated wood shavings

was vanillin followed by dehydroabietic acid (0.26% and 0 .2 %) respectively.

Coniferaldehyde (30mg), stilbene (25 mg), acetovanillone (15 mg) and vanillic acid (As-io)

(15 mg) were the second most abundant compounds respectively. Guaiacol (A b) and

picealactone A were isolated in minor quantities (0.02% and 0.03% )). The chemical structure of

these compounds is shown in Figure 4.2.

Table 4.3 Y ield o f different compounds isolated from heated wood o f P. sylvestris.

C om pound Solvent system M ass obtained(30 g)

Acetovanillone 30% Hexane/ 70% DCM 15 mg (0.05% )

Coniferaldehyde 30% Hexane/ 70% DCM 30 mg (0.1%)

Dehydroabietic acid 30% Hexane/ 70% DCM 60 mg (0.2%)

Guaiacol 100% Hexane 6 mg (0.02% )

Picealactone A 30% Hexane/ 70% DCM 9 mg (0.03% )

Stilbene 30% Hexane/ 70% DCM 25 mg (0.08%)

Vanillic acid 30% Hexane/ 70% DCM 15 mg (0.05%)

Vanillin 30% Hexane/ 70% DCM 80 mg (0.26% )

68

.OH

OHOH

Vanillic acidOH

VanillinAcetovanillone

OH

OH

OH

Abietic acid GuaiacolPicealactone A

HO,

OHHO.

OH

Trans- StilbeneConiferaldehyde(+)-Dehydroabietic acid

Figure 4.2 Compounds isolated from Pinus sylvestris after heat treatment at 120°C.

4.3.5 Synergistic effect of dehydroabietic acid on the antim icrobial activity of vanillin

Initial screening of the fractions derived from a gravity column against SE revealed several

fractions which were combinations of more than one compound, exhibited antimicrobial

activity whereas after purification, the individual compounds had no effect on SE. In order to

investigate the nature of activity of the active mixtures, vanillin and dehydroabietic acid were

purified from active fractions (previously tested against Salmonella) and then tested at

concentrations ranging from 20 to 0.001 mmol against SE. The results presented in Table

4.4 show that SE growth was not affected by pure vanillin at concentrations ranging between

of 20 mmol to 0.001 with more than 100 colony forming units per 20 pi resulting. However,

in combination with dehydroabietic acid, no Salmonella was recovered with up to 0.039

mmol of vanillin. The inhibitory effect o f vanillin/DHAA was still considerable at a

concentration of 0.019 mmol o f vanillin, but dehydroabietic acid alone at a concentration of

20 mmol had no antimicrobial activity on SE.

69

Table 4.4 A comparison o f antimicrobial activity o f vanillin and combinations o f vanillin and dehydroabieticacid on the survival O f SE. (Detection limit =20 cfli/plit)

Salm onella Enteritidis (cfn/plit ± SE)

V anillin (m m ol) (n=3)

D ehydroabietic acid concentration (m m ol)

20 00.0020 0 >10010 0 >1005 0 >100

2.5 0 >1001.25 0 >100

0.625 0 >1000.313 0 >1000.156 0 >1000.078 0 >1000.039 0 >1000.019 30.6 ± 0 .7 >1000.001 >100 >100

0 >100 >100

4.3.6 Effect of low pH on antimicrobial activity of vanillin using citric acid

A review of the literature indicated that vanillin shows stronger antimicrobial activity in an

acidic pH. To evaluate if acidic nature of heat treated wood had contributed to enhanced

antimicrobial activity, citric acid was tested in combination with vanillin. A similar

procedure to that in 4.3.5 was followed and the starting concentration o f the two compounds

was kept the same. The results presented in Table 4.5 showed that citric acid increased the

effect of vanillin very slightly (2- fold maximum) at 20 mmol. However, it had no effect on

SE survival at other tested concentrations of vanillin between 10 to 0.078 mmol.

4.3.7 Antimicrobial activity of commercial vanillin and DHAA

The antimicrobial activity of a combination of commercial vanillin (Fluka, 94752) and

DHAA was set up. The results in Table 4.6 show that 10 and 5 mmol solutions of

commercial vanillin had an inhibitory effect in combination with a 2 0 mmol solution of

DHAA isolated from gum rosin. However, separate solutions of vanillin or dehydroabietic

acids at the same concentrations had no effect on SE survival.

70

Table 4.5 Effect o f citric acid on the antimicrobial activity o f vanillin against Salmonella Enteritidis. (Detectionlimit =20 cfu /lp lit)

Salm onella Enteritidis (cfu/Iit ± SE)

V anillin (m m ol) (n=3)

C itric acid concentration (m m ol)

20.0 00.0020 63.7 ± 3 .3 >10010 >100 >1005 >100 >100

2.5 >100 >1001.25 >100 >100

0.625 >100 >1000.313 >100 >1000.156 >100 >1000.078 >100 >100

0 >100 >100

Table 4.6 Antimicrobial activity o f commercial vanillin alone and in combination with dehydroabietic acid on the survival o f Salm onella Enteritidis. (Detection limit= 20 cfli/plit)

Salm onella Enteritidis (cfu/plit)

V anillin (m m ol) (n=3)

D ehydroabietic acid concentration (m m ol)

20.00 00.0020 0 >10010 0 >1005 0 >100

2.5 >100 >1000 >100 >100

4.3.8 Effect of pH and storage tem perature on antim icrobial activity of commercial

vanillin/DHAA

Stock solutions of vanillin in acetone at a concentration range o f 10, 5, 2.5, 1.25, 0.625 mmol

were combined with DHAA at 20 mmol concentration, at pH 4 and 7 then stored at either

4°C or 25°C for 24 hours. The 10 mmol solutions of vanillin combined with DHAA showed

a bactericidal effect regardless of storage temperature and pH. With lower concentrations of

vanillin (2.5-5 mmol) combined with DHAA, the effect became bacteriostatic to SE, but

again regardless of temperature and pH. However, at lower concentrations of vanillin (1.25

mmol) the bacteriostatic effect was only observed after storage at 25°C and not when

solutions were stored at 4°C (Table 4.7) or when only a very low concentration o f vanillin

(0.625 mmol) was present. At high vanillin concentrations (10 mmol) in the absence o f

DHAA a bacteriostatic effect was observed at pH 4 but not at pH 7. Under, all others

combinations/conditions growth o f SE was observed.

71

T ab le 4 .7 E ffect o f pH and temperature on antimicrobial activity o f on vanillin/ D H A A solution.

T reatm en ts and

storage cond ition

(n=3)

V an illin con cen tration ran ge (m m ol)

W ith D H A A ( 20m m ol) W ith ou t D H A A (20m m ol)

10 5 2 .5 1.25 0.62 10 5 2 .5 1.25 0.62

pH 4 / 4“C BC B S B S G G B S G G G G

p H 4 / 2 5 ”C BC B S B S B S G B S G G G G

p H 7/ 4“C BC B S B S G G G G G G G

p H 7/25" C BC B S B S B S G G G G G G

BC: BactericidalBS: Bacteriostatic G: Growth

4.3.9 Spectrum of antimicrobial activity of the methanol crude extract and isolated

fractions of heated wood of pine

Apart from testing against SE, the methanol crude extract o f heated pine shavings was also

tested against a range o f other organisms: Escherichia coli. Staphylococcus aureus and

Saccharomyces cerevisiae using the bioautographic TEC method described in Chapter 2,

section 2.9.5. In addition, a combination o f vanillin and DHAA was also tested against the

same organisms. The results are shown in Table 4.8. The methanol crude extract o f heated

pine showed antimicrobial activity against all tested microorganisms. The combination o f

vanillin/DHAA also showed inhibitory activity against all tested microorganisms compared

to the pure solutions o f vanillin and DHAA. The pure solution o f stilbene showed some

antimicrobial effect against Staphylococcus, although the mixture o f stilbene/DHAA had

even greater antimicrobial effect.

T able 4 .8 M ean zones o f inhibition (m m ) form ed by active fractions derived from m ethanol crude extract o f

heated w ood o f pine.

T ested stra ins com p ou n d s/ active fraction s(n=3) crud e van illin V /D H A A stilbene S /D H A A D H A A con tro l

Salm onella 10.0±0.3 0.0 14.6±0.5 0.0 0.0 0.0 0.0

E. co li 11.6 ±0.3 0.0 23 .0± 0 .5 0.0 0.0 0.0 0.0

Staphylococcus 16.6± 0.3 0.0 20 .6± 0 .6 8 .0±0.9 18.0±0.0 0.0 0.0

Saccharom yces 8.6 ±0.3 0.0 11.6 ±0.3 0.0 0.0 0.0 0.0

4.4 Discussion

The aim o f the work o f this chapter was to identify potential antimicrobial compounds

responsible for the inhibitory effect exerted by heated wood o f Scots pine against SE.

Therefore, heated wood materials were subject to extraction using different types o f solvents

and the crude extracts then tested against SE. Methanol extracts were found to significantly

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reduce the number o f Salmonella cfu whereas cold and hot water extracts did not. However,

the inhibitory effect o f the methanol extract was only associated with the heat-treated pine

whilst the methanol extract o f air-dried wood had no inhibitory effect on SE numbers. These

results were consistent with the data obtained from a parallel experiment which measured the

recovery o f Salmonella on wood materials which were already water or methanol extracted.

They indicate that that methanol successfully extracts the active compound or compounds

which give heat-treated wood o f pine its antimicrobial activity when in direct contact with

SE.

GC-MS analysis revealed several differences between the chemical composition o f air-dried

and heat-treated pine wood. Heat-treated wood contained at least four new compounds:

vanillin, benzaldehyde, coniferaldehyde and benzoic acid and GC-MS analysis revealed that

vanillin was present in the highest concentration. Preliminary bioassay tests showed fractions

containing vanillin completely inhibited Salmonella recovery from m oist ground perlite.

Vanillin is known to exhibit antimicrobial activity against food-borne bacteria, moulds and

yeasts so it is used as a food preservative in fhiit juice and fruit puree. However, purification

o f the active fractions resulted in a reduction o f this activity. N M R analysis o f the active

fractions revealed that another compound was present in small quantity which was identified

as dehydroabietic acid (DHAA). Further, purification o f the compounds and their individual

bioassays showed the minimum inhibitory concentration o f purified vanillin was 2 0 mmol

when tested against SE and DHAA had no negative effect on SE survival (T able 4.4).

However, when DHAA was tested in combination with vanillin at different concentrations,

the MIC o f vanillin against SE decreased from 20 mmol to 0.019 mmol (Table 4.4).

Previous researches have also reported that DHAA has no antimicrobial activity on Gram

negative bacteria (Savluchinske-Feio et ah, 2006). Also, the minimum inhibitory

concentration o f vanillin against Gram negative bacteria including E. coli and Salmonella

Enteritidis is reported to be over 10 mmol and between 10 to 21 mmol against yeasts

(Fitzgerald et a l , 2004; Vasantha Rupasinghe et a l, 2006). However, the results o f this

study showed a minimum concentration o f 20 mmol o f DHAA in mixture with vanillin at a

concentration range o f between 20 to 0.019 mmol was essential but adequate to inhibit SE

completely. It is known that vanillin shows greater antimicrobial and antioxidant properties

when the pH is acidic (Mourtzinos et a l, 2009). Oxidization o f phenolic compounds during

lignin breakdown increases acidic content o f heated wood. To test the hypothesis that acidic

conditions might enhance the activity o f vanillin, DHAA was replaced with citric acid, but it

73

only increased the effect o f vanillin 2 fold (10 mmol) (Table 4.5). Therefore, the retained

activity of vanillin at low concentrations o f 0.019 mmol in mixture with DHAA may be

explained by the synergistic effect o f the mixture. The synergism between mixtures of

several phenols or abietane type resin acids has been reported previously (Cowan, 1999;

Mourtzinos et a l, 2009; Savluchinske-Feio et a l, 2007). However, the results of the present

study provide the first report o f synergism between an aromatic aldehyde and a diterpenoid

resin acid.

In testing the commercial forms o f aldehydes and resin acids, and finding that the

antimicrobial activity o f their mixture was weakened, it could be argued that other

requirements need to be met for a strong antimicrobial activity to be induced in the

commercial mixtures similar to that obtained in the bioassay o f original fractions. Two

factors are likely to have influenced the magnitude of activity in the original fractions based

on the environment of heat-treated wood and to a similar extent their methanol extracts.

These are (1) low pH caused by the presence o f a high content of organic acids; (2)

temperature effects on the miscibility or solubility o f the compounds. Therefore, mixtures of

the commercial forms were made prior to testing and the effect o f temperature and pH tested.

When vanillin was tested alone, at the same concentration the nature o f activity was only

bacteriostatic at pH 4 and showed no inhibitory effect at pH 7 (Table 4.7). The results also

showed the level o f inhibitory activity o f the mixture was dependent on the vanillin

concentration but it increased only 2 -fold which was less than half o f the antimicrobial

activity of the mixture derived from the methanol crude extract (Table 4.7). In addition, the

nature o f inhibitory activity of the mixture was also dependent on vanillin concentration.

When vanillin was at 10 mmol, the activity o f the mixture was bactericidal at both tested pH

and temperature. Fitzgerald et al (2004a) also reported that the bacteriostatic effect of

vanillin varied depending on the vanillin concentration and type of microorganisms. It is

well accepted that phenolic antimicrobials lose their activity with dilution (Russell, 2003).

However, the mixture derived from heated wood had a bactericidal effect even at the lowest

tested concentration of vanillin (0.019 mmol). This may indicate that the antimicrobial

activity of the mixture is in addition to pH or temperature effects and requires further

investigation. The results of a previous study on resin acid solubility in pulp mill suggest that

these chemicals form association with other organic molecules under acidic condition

(Werker and Hall, 1997). Therefore, the possibility o f such reactions and their significance

on the level of enhanced antimicrobial activity suggests further research in the future.

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Mixtures of the two compounds originating from the crude extract showed an inhibitory

effect not only against SE but also against Escherichia, Staphylococcus and Saccharomyces.

Based on the mean zone of inhibition, the activity of vanillin/DHAA was greater than the

crude extract on each tested species. Maximum inhibition occurred with E. coli and the

minimum effect with Saccharomyces. Both vanillin and DHAA had no activity on any o f the

other tested species when tested alone. However, others have reported antimicrobial activity

of DHAA on Gram positive bacteria including S. aureus (Savluchinske-Feio et al., 1999).

However, when stilbene, another compound derived from crude extract, was tested in

combination with DHAA, it had an inhibitory effect but only on Staphylococcus. Also,

unlike vanillin, purified stilbene alone did have an inhibitory effect on Staphylococcus but to

a lesser extent than the stilbene/DHAA mixture. The antibacterial effect of stilbene on

Staphylococcus aureus has been reported elsewhere (Eloff et a l, 2005), and recently, the

synergistic effect o f stilbene in combination with antibiotics on pathogenic bacteria was

reported for the first time (Kumar et a l, 2012). This highlights the need for further

investigation on the mechanism by which the mixture o f DHAA with other compounds

results in a greater level of antimicrobial activity against different microorganism.

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C H A P T E R 5: E F F E C T O F H E A T E D W O O D O N P. R A M O R U M

5.1 Reasons for concern

Identifying the outbreaks of the disease on Japanese larch in the UK increased concerns that

P. ramorum had the potential to severely affect biodiversity and the shape of the national

landscape throughout the UK. According to Brasier and Webber (2010) this was the first

time that the pathogen had established itself on a commercial plantation tree species,

infecting more than 600,000 mature larch trees (Figure 5.1). Currently, the Forestry

Commission and the private sector are felling all infected larches (Figure 5.2) to reduce the

risk of further damage to uninfected areas. The damage caused to larch has had a significant

economic effect on the value of timber in the UK. It also has raised the possibility that P.

ramorum could become a serious threat to European larch across other countries in Europe.

In 2011, a scientific opinion published by Plant Health Division of the European Food Safety

Authority (EFSA) stated “there are large regions across Europe that are climatically suitable

for spread of Phytopthora ramorum where susceptible hosts are also present” (EFSAP-PLH,

2011).

M '

iFigure 5.1 Dead larch trees infected by Phytophthora ramorum (Source:http://w w w .forestry.gov.uk/pdf/Phytophthoraram orum larchjune2011 .pdf)

5.2 Alternative approaches to control

Since the disease was discovered, researchers have developed control methods to limit the

impact of the disease to the nursery trade and to control its impact on woodland. Measures

include chemical treatment of plant stocks showing limited infection, irrigation water

disinfestation and soil disinfestation using chemical compounds. Such management

strategies have been successful in preventing significant damage to nursery plants. Despite

76

this, none of these treatments are guaranteed to eliminate the disease. Moreover, these

treatments are costly to the nursery trade due to the need to destroy infected stock (Frankel,

2008). Effective existing strategies are even more limited for infestations in the wider

environment. In heavily contaminated areas in the UK, trees are cut and logs transported to

licensed mills to be debarked and processed. Where clear cutting is not an option, removing

foliar hosts is carried out to reduce the spread of the disease to other trees. As the complete

clearance of foliar hosts in the wider environment is not economically viable, alternative

strategies are needed to limit inoculum levels and reduce the movement of inoculum from

contaminated litter to standing tree species.

Figure 5.2 Larch trees infected by Phytophthora ramorum, C rym ant, near N eath (Source: w w w .telegraph.co.uk/earth/countrvside/8298946)

One alternative strategy, covering the litter using a layer of woodchips has been practiced,

acting as a physical barrier to reduce the mobility of inoculum (Goheen et al., 2012).

However, using woodchips with antimicrobial activity potentially has more benefits than use

only as a physical barrier. Research on the chemical characteristics of woodchips used from

different tree species indicates the significance that different species may have on survival of

P. ramorum. For example, the survival of P. ramorum was investigated on woodchips of

rhododendron under natural conditions in a private garden in the Netherlands where the

disease was already presented (Boogert and Kessel, 2004). The study was undertaken in 16

plots over a period of 18 months. For each plot, 300 grams of woodchips were layered over

lm “ areas, 1 month prior to sampling in June 2004 when samples were taken to be tested for

77

presence of P. ramorum. Positive results were obtained throughout the study at each time of

sampling (variation between 1 to 10 samples). In contrast, woodchips from several western

conifer species and the identified compounds in the woodchips were found to have strong

antimicrobial activity toward P. ramorum (Manter et a l, 2006). The author suggested the

benefit of using antimicrobial woodchips as a part of an integrated programme to minimize

spore distribution in the area with higher human activities (Davidson et a l, 2005a; Tjosvold

et a l, 2 0 0 2 b).

Heating o f wood has been shown to be an effective method to improve its resistance against

fungal attacks. Heating to a temperature of 200°C, leads to wood becoming resistant against

blue stain, white rot and brown rot fungi (Kamdem et a l, 2002; Savluchinske-Feio et a l,

2007). Depending on the fungus and wood species, decaying o f wood can be described as the

depolymerisation or modification of the main wood components resulting in the release of

energy and metabolites for microbial growth. In the first instance, biological resistance of

heated wood can be explained by the changes caused by heating which ensure it is no longer

a favourable habitat for fungal growth. However, extensive research on the effect o f heat

treatments on biological resistance has shown the formation of new chemicals in thermally

modified wood contributes towards the toxicity o f heated wood for many fungi and bacteria.

In the earlier Chapters of the present study, chemical analysis of heated wood o f pine showed

the existence of potential antimicrobial compounds. Research reported in this chapter

therefore aims to evaluate the potential of antimicrobial activity of heat treated wood o f pine,

larch and rhododendron using the heating regime developed in Chapter 1 to control P.

ramorum. Initially, woodchips of pine were tested by direct contact with P. ramorum

progagules. In the later stages of the study, woodchips o f rhododendron and larch, the two

main hosts of P. ramorum in the UK, were then tested for their antimicrobial activity against

P. ramorum. Chemical analysis o f all three tested species was carried out and different

chemicals isolated and tested individually against P. ramorum to assess the active chemicals.

Finally, to evaluate the strength and magnitude of the enhanced activity under natural

conditions, field trials were set up in two different locations in south west England where P.

ramorum was present and the heated woodchips materials regularly monitored for the

presence of pathogen over a period of 6 month.

78

5.2 Experimental

5.2.1 Micro cosm trails

The antimicrobial activity of woodchips derived from pine, rhododendron and Japanese larch

was tested in a number of different trials. Heat-treated and air-dried woodchips were

prepared as described in Chapter 2 (2.10.1). Details o f test isolates of P. ramorum are also

given in Chapter 2 (2.10.3).

5.2.1.1 Effect of larch, pine and rhododendron heated woodchips on the survival of

Phytophthora ramorum zoospores

To assess the effect o f heat treated woodchips on growth and survival o f P. ramorum,

woodchips were obtained from the selected species using ten replicates for each treatment.

For each replicate, Ig of woodchips was placed in a Universal bottle, sealed and autoclaved

for 15 minutes at 120°C. Each was then inoculated with 1 ml of a spore suspension

(lO'^zoospores mf^) of the test P. ramorum isolates. Inoculated woodchips were then

incubated at 20°C for 3 weeks, then Ig of the woodchips of each treatment was placed on

Phytophthora selective medium (SMA) to monitor the survival of zoospores. Survival was

expressed as the number of treatments with positive growth of the pathogen from the

woodchips into the agar.

To detect the ability o f the pathogen (in the form of zoospores) to germinate and form

sporangia on the heated woodchips, one extra set of the same treatments was prepared and

received 1ml of the same spore suspension and an additional 1 ml o f sterile water. The

treatments were left under continuous light to induce sporangia for 2 weeks at 20°C. All the

bottles then received 1 0 ml of sterile water and were shaken well ( 2 1 bottles for each isolate)

before they were transferred to 4°C for an hour and moved back to ambient temperature for

45 minutes so zoospores were released from sporangia. The water content from each

universal bottle was transferred into a 25 ml sterile plastic bottles, 10 drops of 1 pi from each

bottle was placed on a glass slide. Using a microscope, the drops were then examined for

chlamydospores, zoospores and sporangia.

5.2.1.2 Effect of incorporating heated woodchips made from larch into soil infected with

P. ramorum

An experiment was designed to assess the potential of incorporating heated larch woodchips

to inhibit P. ramorum inoculum when combined. Two isolates BRCOl and P I578 were used

in the experiment. Heated and air-dried woodchips o f larch were ground to fine particles to

79

ensure a high level of interaction with soil. Soil free from P. ramorum was taken from a local

woodland (Alice Holt Forest, Famham, Surrey) and mixed with ground larch woodchips in

ratios of 25% and 75% (w:w) inside plastic bags. Each treatment received sterile water to its

water holding capacity, was sealed and left to settle at 25°C for 7 days. Plastic bags were

agitated and then the mixture was stirred every two days. A zoospore suspension which was

made as described in 2.10.3.1 (lO^zoospore mf^) and each bag received 10 ml o f the

suspension and was thoroughly mixed manually. Perlite was incorporated with soil in the

same ratios to act as controls. After the addition of the spore suspension, the bags were

sealed and stored at room temperature (20°C) for 4 weeks; thenl g o f the mixture was

removed from the bag, diluted with 1 0 ml sterile water and 1 ml o f the suspension plated onto

SMA. The same process was repeated after 8 weeks o f storage. The SMA plates were

incubated for 7 days at 20°C and any colonies o f P. ramorum were counted (cfu g" )̂. The

data were analysed by a one-way Analysis o f Variance ANOVA, GraphPadPrism, version

InStat3. The Tukey Post Analysis Test was performed to assess the least significance

difference between means (P<0.05). Survival of P. ramorum in the remaining mixtures in the

plastic bags was also tested.

5.2.1.3 Effect of incorporated heated woodchips made from larch on the survival of P.

ramorum chlamydospores

Apart from zoospores, P. ramorum produces chlamydospores that are considered to be long-

lived and resilient resting spores (Linderman et al., 2006). Chlamydospores of P. ramorum

are able to survive in soil for a long period o f time. To assess the inhibitory effect o f heated

woodchips when incorporated into the soil on the survival of chlamydospores, pre-colonized

leaf disks of rhododendron containing chlamydospores of P. ramorum were incorporated into

woodland soil (see above) which was then mixed with 75% heated larch woodchips.

Controls consisted of 100% soil and 100% heated woodchips. Treatments were compared

with an inert soil amendment consisting of 25 and 75% perlite. For each treatment, 12 leaf

disks previously colonized with isolates BRCOl and P I578 were used. The leaf disks were

recovered from the pots 4 weeks and 12 weeks after incorporation, rinsed with sterile water

and then plated onto SMA. Plates were incubated for at least 3 weeks at 20°C before each

disk was assessed for growth of P. ramorum. When the assays were completed, to assess if

P. ramorum had established in the soil itself, the soil from each pot was placed in another

container, flooded with sterile water and baited with 12 fresh rhododendron leaf disks. After

5 days incubation at 20°C, the bait disks were plated on SMA to check for any outgrowth o f

80

p. ramorum. The data were analysed by one-way ANOVA, GraphPadPrism, version InStatS.

The Tukey Post Analysis Test was performed to assess the least significance difference

between means (P<0.05).

5.2.2 In vitro assays

5.2.2.1 Assessment of Minimum Inhibitory Concentration (MIC) of crude extracts

against P, ramorum zoospores

For the assessment of the Minimum Inhibitory Concentration (MIC) o f the methanol crude

extracts derived from air-dried and heated wood were tested against two P. ramorum isolates

BRCOl and P I578. Concentrations o f 50, 25, 12.5, 6.25 and 3.12 mg o f extract per ml in

acetone were prepared. Half strength carrot broth was also prepared to provide a growth

medium for P. ramorum and was dispensed into each well of the assay plate and the acetone

evaporated in a fume hood. After evaporation, each well was inoculated with 5 pi of a fresh

zoospore suspension of P. ramorum containing 10"̂ zoospores ml"\ Each well contained a

final concentration range of 50, 25, 12.5, 6.25 or 3.12 mg of crude extract per ml o f carrot

broth. Plates were incubated for 24 hour before 20pi of the broth was removed and plated

onto SMA. The SMA plates were incubated at 20°C and scored as positive if growth o f P.

ramorum was detected. Each treatment was replicated three times.

5.2.2.2 Effect of crude extracts derived from heated and air-dried wood on

chlamydospore germination of P. ramorum

An experiment was set up to assess the effect o f the methanol crude extract derived from air-

dried and heated wood of pine, larch and rhododendron against P. ramorum chlamydospores

from isolates BRCOl and P I578. To test the effect of the different methanol extracts, each

crude extract was made in acetone. The test used leaf disks of rhododendron pre-colonized

with chlamydospores of P. ramorum. To expose the chlamydospores to the crude extract, a

six well plate was filled with 1 ml of sterile water in each well. Subsequently 1 ml o f extract

was pipetted into each well and the plates placed in a fume hood for 16 hours to allow the

acetone to evaporate giving a crude extract concentration in water of 10 mg m l '\ For each

treatment, two wells were used, each containing six pre-colonized leaf disks. To ensure good

contact between the chalamydospores and the extract, plates were incubated for 48 hours on a

platform shaker at ambient temperature. After 48 hours incubation, disks were removed from

the solution, rinsed in sterile water for Ih before being placed onto SMA. The SMA plates

were incubated for 3 weeks and the number of disks (average out of 6 disks from each o f two

wells) showing no outgrowth of P. ramorum, were calculated.

81

5.2.2.3 M IC of chemicals in crude extracts derived from heated pine wood against

hyphae, zoospores and chlamydospores of P. ramorum

The Minimum Inhibitory Concentration (MIC) of the compounds found in the crude

methanol extract derived from heated pine wood was assessed. The compounds included

coniferaldehyde, acetovanillone and vanillin which were tested against mycelia, zoospores

and chlamydospores of two isolates of P. ramorum, BRCOl and P I578. A two fold dilution

series of each compound (range from 20 to 0.009 mmol) in DMSO (dimethyl sulfoxide) was

prepared and dispensed in multi-well plates containing zoospores, chalamydospores and

mycelia which were prepared as described in 2.10.3.1. Each treatment was replicated three

times. Plates were incubated at 20°C for 24 hours for zoospores and 48 h for chlamydospores

and mycelia. After incubation the content of each well was plated onto SMA plates and

incubated for 5 days before assessing growth of P. ramorum. The minimum concentration at

which no P. ramorum colonies developed was recorded as the MIC.

To assess if resin acids lowered the MIC o f the various compounds, the assay was repeated

with combinations of the compounds plus dehydroabietic acid tested against zoospores of

three P. ramorum isolates (BRCOl, P1578 and P2460). The dehydroabietic acid

concentration was kept at 20 mmol. A further test was set up to assess if different resin acids

(dehydroabietic acid, abietic acid and pimaric acid) differed in their ability to increase the

activity of acetovanillin, coniferaldehyde and vanillin.

5.2.3 Field studies

5.2.3.1 Largin Wood field evaluation

Experimental assays described above indicated that heat treated woodchips particularly of

larch and pine, could be inhibitory to zoospores and chlamydospores of P. ramorum when

tested in the lab. Therefore, trials were designed to assess whether heat treated woodchips of

natural hosts of P. ramorum (rhododendron and larch) would inhibit the growth o f P.

ramorum under conditions in the field with variable inoculum loads. In November 2011, a

field study was conducted at Largin Wood, Forestry Commission, forest to assess the ability

of heated woodchips to inhibit the establishment of P. ramorum in soil and the litter layer.

The location was chosen as it was likely that a source o f natural P. ramorum inoculum

existed. Prior to placement of wood samples, several foliage and soil samples were collected

from three different spots at the site and were baited using rhododendron leaf disks. All three

spots proved P. ramorum positive. The selected points were labelled and wood samples were

prepared and placed on the litter in the site (Figure 5.3).

82

To prepare the sample, woodchips of rhododendron, larch and pine were wrapped in

aluminium foil and heated for 72 hours at 120°C in a dry oven. The heated samples of each

wood species were then split into 30 batches o f 150 g o f woodchips. Each batch was sealed

in a fine mesh plastic bag and labelled. The same number of air-dried woodchip samples of

pine, larch and rhododendron were also bagged in 150 g batches and sealed. All the bags

were transferred to the field and 1 0 bags o f each treatment (heated v& air-dried) of each wood"

species were placed on the litter in each spot (60 bags) (Figure 5.3). A set o f 18 sample bags

( 6 from each spot) were collected from the field on a monthly basis; for analysis each bag

was transferred to a plastic container and baited using rhododendron leaf disks which were

placed onto SMA after 5 days. The numbers o f leaf disks with positive outgrowth of P.

ramorum (n=30) were recorded for comparison. The data were analysed by a one-way

ANOVA, GraphPadPrism, version InStat3. The Tukey Post Analysis Test was performed to

assess the least significance difference between means (P<0.05).

5.2.3.2 Druid’s Hill field evaluation

A second experiment was set up between May and December 2012 to assess the inhibitory

effect of heat treated larch alone. The principle of this study was based on the fact that

woodchips or litter can become infected with P. ramorum when exposed to aerial inoculum

coming from infected foliar hosts. A5 size seed trays with drainage holes in the bottom were

filled with a 5cm layer o f wood chips made from heated larch woodchips. Air-dried

rhododendron wood chips were used as a control. Whole rhododendron leaves were used as

baits and placed on the top o f the woodchips. The trays were also covered with a fine mesh

net to protect them from animal damage. To minimise the risk of inoculum being washed off

by heavy rain, each tray was placed in a larger seed tray (without any drainage holes). In this

study site, the inoculum came from infected foliar o f R. ponticum and woodchips were placed

underneath infected rhododendron bushes (Figure 5.4).

83

r.

Figure 5.3 Field site in Largin W ood.

(a) landscape view o f the field site; (b) and (c) Nylon net bags containing w oodchips on site; (d) bagged sam ples o f w oodchips prior to analysis in the lab.

Samples were taken directly from the trays, and from the water that collected in the bottom

tray. To detect the presence of any viable P. ramorum, samples were baited with

rhododendron disks or subjected to RT-PCR for the detection of mRNA.

No P. ramorum could be detected in any of the treatments (air-dried or heat-treated wood

chips of larch and rhododendron) during May, June, July and August 2012. Test methods for

detection were both baiting and detection of live cells of P. ramorum using an mRNA based

RT-PCR assay (Chimento et al., 2011). Samples were negative (24 samples of larch and

rhododendron tested in July and all 36 tested samples in November).

84

Figure 5.4 Exam ple o f trays w ith w ood chips placed underneath a rhododendron bush at D ru id ’s H ill, Cornwall

(a) at the tim e o f set up in M ay 2012, (b) in D ecem ber 2012.

To determine the minimum detection limit of the RT-PCR assay (Chimento et al., 2011), a

dilution series of zoospores from the isolate of P. ramorum originating from foliage at the

survey site was prepared. After vacuum filtration, a concentration range of 1, 10, 100, 1000,

10,000, 100,000 and 1,000,000 zoospores m f’ in sterile water was made. The zoospore

concentrations of 1 , 1 0 and 1 0 0 zoospores m f’ were adjusted using a microliter pipette by

following the method described by Xu and Ko (1998). Five drops of the spore suspension

(each 1 pi) were placed on a glass slide and the number of spores was counted using a

microscope as a check. The drops were then recovered using a micropipette and transferred

to RNase free tubes. Concentrations above 100 zoospores m f’ were adjusted to the mean

value of four counts of spore suspensions made by a haemocytometer. RNA was extracted

from the samples using RNeasy Plant MiniKit (Qiagen Inc, Valencia, CA, USA) for total

RNA isolation from plants and filamentous fungi, following the manufacture’s protocol.

After elimination of genomic DNA, reverse transcription was performed by two-step RT-

PCR using QuantiTect® Reverse Transcription kit (Qiagen) following the manufacture’s

procedure and RT Primer Mix (Qiagen). Then, cDNA was amplified using

ACPramF/ACPramR primer set. PCR was performed in a total volume of 25 pi containing

6.26 pi cDNA, with 12.5 pi of SYBER® Green Mastermix containing 0.5 pmol of each

primer. Real-time amplification was carried out in a Real Time thermal cycler (LightCycler®

480 II) using the following program: one cycle at 50°C for 10 minutes, one cycle at 95°C for

3 minutes, 35 cycles at 95°C for 15 second and 63°C for 1 minutes. Ramp rate was 3.3°C/s

heating and 2°C. PCR amplification with Ct values (threshold cycle) lower than 31 were

considered positive.

85

5.2.4 Chemical analyses

5.2.4.1 Chemical analysis of heated larch, pine and rhododendron wood

GC-MS analysis of the methanol crude extracts of heat-treated pine, larch and rhododendron

(1 mg ml'* methanol) was carried out to compare the chemical constitutes (Figure 5.9). All

three crude extracts were dissolved in methanol as described in Chapter 2 (2.9.2) and were

subjected to Gas Chromatography Mass Spectrometry.

5.2.4.2 Chemical analysis of discoloured bark of a Japanese larch

When bark of Japanese larch is infected by P. ramorum, the phloem tissue undergoes a

number of colour changes that appear to be the response of the tree as it defends itself against

the pathogen (Figure 5.5). To assess what chemical changes in the bark could be associated

with the alternation in colour; a GC-MS analysis of the different bark samples was

performed. The analysis used DCM crude extracts of bark at different stages of

discolouration following invasion by P. ramorum. These consisted of: white tissue not yet

invaded; red tissue indicating a response to P. ramorum but usually LTD negative; dark

brown tissue, LTD positive and P. ramorum could usually be cultured from this tissue. Apart

from these samples, a DCM crude extract of the white phloem tissue from a healthy tree was

included for comparison.

f iFigure 5.5 Bark (phloem ) o f a Japanese larch tree show ing different stages o f discolouration, (a) white (b) red and dark brown tissue formed as a response to invasion by P. ramorum.

86

5.3 Results

5.3.1 Microcosm studies

5.3.1.1 Evaluation of woodchips derived from larch, pine and rhododendron

The experimental work was set up to test the hypothesis that heating woodchips , of pine larch

and rhododendron would make them inhibitory to the pathogen P. ramorum. Two isolates of

P. ramorum, BRCOl and P I578 were used in all experiments. Survival o f P. ramorum

zoospores applied to heated (72 hours at 120°C) and air-dried woodchips was assessed using

the methods described in 5.2.1.1, and the results are shown in Table 5.1. All three types of

heat-treated woodchips completely inhibited the recovery of zoospores of both test isolates

compared to the air-dried wood of the same species (P<0.001). Heat-treatment affected the

zoospore production on all three wood types of P. ramorum compared to air-dried wood. All

inoculated air-dried wood treatments yielded colonies o f P. ramorum after plating whereas P.

ramorum could not be isolated from any of the heated wood treatments and the treatment did

not allow any reproduction of the fungus.

Table 5.1 Survival o f P. ramorum zoospores inoculated on air-dried and heated woodchips.

W oodchip treatm ent P. ram orum positive (M ean ± SE)

Z oospore presence

B R C O l P1578 B R C O l P1578Pine : air-dried 10 ± 0 .0 9.0 ± 0.7 Positive Positive*Larch: air-dried 8.2 ± 0.3 9.7 ± 0.0 Positive Positive*Rhododendron: air-dried 10 ± 0 .0 10 ± 0 .0 Positive PositivePine: heated 0.0 0.0 Negative NegativeLarch: heated 0.0 0.0 Negative NegativeRhododendron: heated 0.0 0.0 Negative Negative

*Presence o f chlamydospores

Table 5.2 Effect o f incorporation rate o f heated larch woodchips into soil on the survival o f two isolates o f P. ramorum, 3 days and 8 w eeks after inoculation (n=3).

Treatm ent

Z oospore assay D isk assayB R C O l ( cfu/g) P1578 (cfu/g) B oth isolates

3 days 8 w eeks 3 days 8 w eeks 8 w eeks

100% larch 0.0“* 0.0“ 0.0“ 0.0“ N egative

75% Larch/ 25% Soil 0.0“ 0.0“ 0.0“ 0.0“ Negative

25% Larch/ 75% Soil 0.0“ 0.6 ± 0 .1 “ 0.0“ 0.5 ± 0 .1 “ Positive

75% Perlite/25% Soil 3.8 ± 0.7^ 1.0 ± 0 .0 '’ 2.1 ± 0 .3 '’ 1.2 ±0.1^ Positive

25% Perlite/75% Soil 5.6 ± 0.8'’. 2.1 ± 0 .2 ' ’ 4.4 ± 0.5° 2.0 ± 0.0° Positive

100% Soil 6.7 ± 0.2° . 4.2 ± 0.4° 5.0 ± 0.2° 3.5 ± 0.6° Positive

Significance P<0.001 P<0.001 P< 0.001 P<0.001

*Different letters represent significant differences between means

87

5.3.1.2 Effect of incorporating heated woodchips into soil infested with zoospores

This experiment was set up as described in 5.2.1.2, to assess the effect o f incorporating

heated larch woodchips into soil artificially infested with P. ramorum inoculum.

Phytophthora ramorum was not recovered from treatments containing 100% and 75% heat-

treated larch woodchips after 3 days and 8 weeks. In contrast, both isolates were recovered

frequently from the soil mixed with perlite (Table 5.2). It was also recovered at low levels

from mixtures that contained only 25% larch woodchips.

Results of baiting the treatments gave P. ramorum positive for heated larch woodchips mixed

at 25% with perlite and 75% with soil. Heated larch wood at ratios of 75% and 100% were

P. ramorum negative.

5.3.1.3 Effect of incorporated heated larch woodchips on the survival of chlamydospores

Recovery of P. ramorum chlamydospores was assessed based on outgrowth of pathogen from

pre-colonized leaf disks of rhododendron which were incorporated into a homogenized

mixture of heated larch wood with soil. The experiment was assessed after 4 and 12 weeks

incubation (Table 5.3). Recovery o f the pathogen occurred with only 2.7% of leaf disks from

P. ramorum P I578 treatment and 5.7% from BRCOl treatment after 3 days incubation. In

contrast, 8 6 % of leaf disks from P. ramorum BRCOl and 91.6% form P. ramorum P I578

were recovered from the treatment pots containing 75% perlite with 25% soil. After 4 weeks,

none of the recovered leaf disks showed outgrowth of P. ramorum from the 75% and 100%

larch woodchips treatments, while over 90% of leaf disks recovered from 75% perlite/soil

treatments were P. ramorum positive. However, 100% of leaf disks recovered from 100%

soil treatments were P. ramorum positive, both 4 and 12 weeks after incubation.

Table 5.3 Effect o f heated woodchips o f larch on the germination o f lea f disks o f rhododendron.(L eaf disks containing chlamydospores o f two isolates o f P. ramorum: B R C O l, P1578, 4 and 12 w eeks after

inoculation (n=12)

T reatm entA verage percentage o f lea f disks w ith outgrow th o f P. ram orum

B R C O l P15784 w eeks 12 w eeks 4 w eeks 12 w eeks 24 w eeks

100% Larch 0.0 0.0 0.0 0.0 N egative75% Larch/ 25% Soil 5.7% 0.0 2.7% 0.0 Negative75% Perlite/25% Soil 86% 91.6% 91.694 94.4% Positive100% Soil 100% 100% 100% 100% Positive

88

5.3.2 In vitro assays

5.3.2.1 Assessment of minimum inhibitory concentration (MIC) of crude extracts

against zoospores

Crude methanol base extracts were made from air-dried and heat treated woodehips of pine,

lareh and rhododendron. The inhibitory effeet was tested against spores and myeelia

obtained from two isolates of P. ramorum. The methanol crude extracts o f the air-dried pine,

larch and rhododendron wood had no inhibitory effect on isolate BRCOl at any o f the

concentrations tested while isolate P I578 was inhibited by air-dried wood extracts from both

pine and lareh at a MIC o f 25 mg ml"\ In contrast, the methanol erude extracts derived from

heated pine, lareh and rhododendron all showed an inhibitory effect against both of the P.

ramorum isolates tested. The lowest MIC was reeorded for crude extraets from heated pine

and larch which both had a MIC of 6.25 mg ml'^ against P. ramorum P1578, a MIC o f 6.25

mg ml'^ for heated larch extract and a MIC o f 12 mg ml'^ for heated pine extraet against P.

ramorum BRCOl. Methanol extracts derived from heated rhododendron wood had MICs of

25 mg ml'^ against P. ramorum P1578 and a MIC o f 50 mg ml'^ against P. ramorum BRCOl

(Table 5.4).

Table 5.4 Minimum inhibitory concentration o f the methanol crude extract (mg ml ') o f air-dried and heated wood o f pine, larch and rhododendron (n=3).

W ood extractTreatm ent and P. ram orum isolates

H eat-treated A ir-driedB R C O l P1578 B R C O l P1578

Pine >12.5 >&25 0.0 >25Larch >&25 >&25 0.0 >25Rhododendron >50 >25 0.0 0.0

5.3.2.2 Effect of crude extracts derived from heated and air-dried wood on

chlamydospore germination

The methanol crude extracts o f heat-treated wood of larch, pine and rhododendron at a

eoncentration of 10 mg ml'^ significantly (P<0.001) reduced germination o f ehlamydospores

of both isolates of P. ramorum by more than 90% eompared with similar extraets from air-

dried wood. The average number of leaf disks eolonised with ehlamydospores from which P.

ramorum could be isolated was much higher when the extract came from air-dried wood,

regardless of the plant species (Table 5.5).

89

Table 5.5 Effect o f m ethanol crude extracts o f air-dried and heated w ood o f pine, larch and rhododendron on germ ination o f pre-colonized lea f disks o f rhododendron with chlam ydospores o f tw o isolates o f P. ramorum. Data presented represent num ber o f lea f disks (out Of 6) that failed to yield any P. ramorum in two replicates o f each treatm ent.

IsolatesM ean num ber o f lea f disks w ithout outgrow th o f P. ram orum

H eat treated wood A ir-dried w oodPine Larch Rhododen Pine Larch Rhododen

P1578 5.75 ± 0 .2 6.00± 00 5.25± 0.2 1.0 ± 0 .7 0.5 ± 0.2 0.25 ± 0 .2

B RC O l 5.5 ± 0.5 5.5 ±0.2 5.0 ± 0 .4 0.0 0.0 0.0

Effect o f Isolate NS NS N S NS NS N SEffect o f W ood type NS NSEffect o f heat treatm ent P<0.001

5.3.2.3 Minimum Inhibitory Concentration (MIC) of different chemicals found in crude

extracts derived from heated pine wood

Pure compounds were 2-10 times more active against zoospores, chlamydosores and myeelia

of P. ramorum than the erude extract (Table 5.5). Coniferaldehyde was the most active

compound with a MIC of 1.7 mg m f' against myeelia and chlamydosores of P. ramorum and

a MIC of 0.15mg m f’ against zoospores (Table 5.6). Aeetovanillone and vanillin had similar

anti-microbial properties with MICs of 3.4-6 . 6 mg m f’ against myeelia and ehlamydospores

and MICs of between 1.5 and 1.6 mg m f’ against zoospores. All compounds (including the

crude extract) showed similar activities against both P. ramorum isolates tested (Table 5.6).

Table 5.6 M inim um inhibitory concentration (m g m l ' ) o f the com pounds derived from heated wood o f pine on m yeelia, zoospores and chlam ydospores (in colonized lea f disks) o f tw o isolates o f P. ramorum.

C om poundsM inim um inhibitory concentration (m g ml ')

M yeelia Zoospores C hlam ydosporesIsolates BR C O l PI 578 B R C O l PI 578 B R C O l P1578

Crude extract 25 25 6 2 5 6 2 5 10 10

Aeetovanillone 6.6 3.3 1.6 1.6 6.6 3.3

Coniferaldehyde 1.7 1.7 0.15 0.15 1.7 1.7

Vanillin 3.4 3.4 1.52 1.52 3.4 3.4

(a) (b)

Figure 5.6 Exam ples o f an assay plate (96-well plate) after the chem ically treated zoospores are plated on SMA (a), and (b) an enlarged picture o f normal grow th o f P. ramorum in sterile w ater (control).

90

When combined with 20mmol dehydroabietic acid (DHAA), the MIC of coniferaldehyde and

aeetovanillone was reduced 8 -fold, and when combined with vanillin, the MIC was reduced

4-fold (Table 5.7).

Table 5.7 Minimum inhibitory concentrations o f aeetovanillone, coniferaldehyde and vanillin, with and without 20 mM dehydroabietic acid (DHAA) tested against zoospores o f P. ramorum (n=3). •

C om poundsM IC m g ml ' against P. ram orum zoospores

B R C O l P1578 P2460Aeetovanillone 1.6 1.6 1.6Aeetovanillone + 20mM DHAA 0.2 0.2 0.41Conifer aldehyde 0.15 0.15 0.15Conifer aldehyde + 20mM DHAA 0.02 0.02 0.02Vanillin 1.52 1.52 1.52Vanillin + 20mM DHAA 0.38 0 3 8 0.38

The compound combinations tested against the two isolates of P. ramorum resulted in similar

MIC levels (Table 5.7).

In separate assays when aeetovanillone, coniferaldehyde and vanillin were combined with

both dehydroabietic acid (DHAA) or abietic acid (AB) at 20 mM the MIC decreased 4 to 16

fold, whilst the addition of pimaric aid had no effect on the MIC of the tested compounds

(Table 5.8).

Table 5.8 Minimum inhibitory concentration o f aeetovanillone, coniferaldeyde and vanillin with or without either 20mM o f abietic, dehydroabietic or pimaric acid tested against zoospores o f P. ramorum isolate B R C O l. Three replicates o f each concentration were made and experiment was repeated three times.

C om poundsM IC m g m l ' against zoospores o f P. ram orum

+W ater +A A +D H A A +PAAeetovanillone 1.6 0.1 0.2 1.6Conifer aldehyde 0.1 0.02 0.02 0.11Vanillin 1.5 0.38 0.38 1.5

5.3.3 Field studies

5.3.3.1 Largin Wood evaluation

The laboratory assays described above indicated that heat treated woodchips, particularly

those of larch and pine, could be inhibitory to zoospores and chlamydospores of P. ramorum.

Therefore, trials were designed to assess whether heat treated woodchips of natural hosts of

P. ramorum (rhododendron and larch) would inhibit the growth o f P. ramorum under

conditions in the field with variable inoculum loads.

In November 2011, a field study was conducted at Largin Wood, Forestry Commission

Forest Cornwall, to assess the ability of heated and air-dried woodchips to inhibit the

91

establishment of P. ramorum in soil and the litter layer. The location was chosen as a source

o f natural P. ramorum inoculum. However, no P. ramorum was isolated from either air-dried

or heat treated wood chips o f pine, larch or rhododendron 4, 8 and 12 weeks after their

placement on the site in November 2011 (Table 5.9). After 20 weeks, P. ramorum was

isolated from the air-dried woodchips of rhododendron which demonstrated that natural

inoculum was present, but not from any o f the other treatments. The difference between the

treatments was not statistically significant. After 52 weeks on the site (November 2012),

there was more than 2-fold increase in the number of baiting disks infected with P. ramorum

recovered from air-dried larch and pine wood chips compared with heated wood chips

(P<0.001). P. ramorum was isolated from both heat treated and air-dried rhododendron

wood chips after 52 weeks, but although fewer infected bait disks were associated with the

heat treated woodchips the treatments, were not significantly different.(Table 5.9).

Table 5.9 Effect o f heat treatments on survivals o f T. ramorum on woodchips o f larch, rhododendron and pine. Treatments were baited separately with 30 rhododendron lea f disks per sample.

Sam pling intervals

4, 8 ,1 2 w eeks 20 w eeks 52 w eeks

17.12.11 18.1.12 17.2.12 1 9 .4 .1 2 7.11.12

Treatm ents H eated A ir-dried H eated A ir-dried H eated A ir-dried Significance

Larch 0.0 0.0 0.0 0.0 3.1 ± 2 .1 13.5 ± 5 .0 P< 0.001

Pine 0.0 0.0 0.0 0.0 3.8 ± 2 .5 16.1 ± 2 .9 P< 0.001

Rhododendron 0.0 0.0 0.0 2.8 ± 1 .8 8.5 ± 3.3 16.1 ± 5 .0 N S

5.3.S.2 D ruid’s Hill field evaluation

No P. ramorum was detected in any of the treatments (air-dried and heat-treated wood chips

of larch and rhododendron) during May, June, July and August 2012. Detection o f live cells

of P. ramorum by performing mRNA based RT-PCR assay (Chimento et a l, 2012) was

negative for all 24 samples of larch and rhododendron tested in July and all 36 samples tested

in November. To determine the minimum detection limit of the RT-PCR assay, a dilution

series o f zoospores from the isolate of P. ramorum originated from foliage o f the field site

and cultured on carrot agar was prepared. A concentration range of 1, 10, 100, 1000, 10000,

100,000 and 1,000,000 zoospores mf^ in sterile water were tested. The minimum detection

limit was 10"̂ zoospores of the tested isolate per ml for the tested RT-PCR assay.

92

5.3.4 Chemical analyses

5.3.4.1 Chemical analysis of heated larch, pine and rhododendron wood used in the field

GCMS analysis o f the methanol crude extracts o f heat-treated pine, larch and rhododendron

(Im g ml'^ methanol) was carried out to compare the chemical constitutents (Figure 5.7).

Spectra o f all three crude extracts had peaks for vanillin at 13.7 minutes, vanillic acid at 15.8

minutes, and syringaldéhyde at 16.9 minutes. The peak for vanillin was considerably higher

in the crude extract o f pine and larch compared to rhododendron. One peak at 17.7 minutes,

identified as coniferaldehyde (NIST library), was only present in larch and pine extracts. In

contrast, benzoic acid (18.67 minutes), syringic acid (18.73 minutes) and sinapaldehyde

(20.34 minutes) were only present in the crude extract o f rhododendron (Figure 5.7).

5.3.4.2 Chemical analysis of discoloured bark of a Japanese larch

The analysis o f the DCM crude extracts revealed that prior to detection o f the pathogen in

invaded bark, white phloem tissue showed an abundance o f resin acid 2 (2 4 .8 minutes) and

much lower amounts o f resin acid 1 (24 minutes) compared with the phloem from the

healthy control (Figure 5.8 a, b). However, in the crude extract o f the red-discoloured bark,

resin acids 1 and 2 were both present in considerable quantities, whereas in the brown tissue

resin acid 2 was at levels similar to the control (Figure 5.8 c, d). Due to trace quantity o f

resin acids, they were identified firom a chromatogram o f a spiked sample o f DCM crude

extract with 10 mg o f synthetic DHAA and AB. The resin acid number 2 was identified as

dehydroabietic acid (A2 1 ) and resin acid number 1 was identified as methyl abietate (A2 0 ), a

derivative o f abietic acid. It was also striking that particularly the red discoloured tissue had

raised levels o f many other compounds compared with the control and white coloured

phloem tissue. The monoterepenes, a-pinene (6.14 minutes) and 3-carene (7.58 minutes)

both were only present in the extract o f red and dark brown phloem which were formed

during and after pathogen invasion (A2 2 and A 2 3 ).

93

(a)

I K ; t 41 ffsl I \ i t: if T?.

1 vanillin2 vanillic acid3 syringaldéhyde4 coniferaldehyde5 benzoic acid6 syringic acid7 sinapaldehyde

-OCD 12CD 1400 -BOD 1BOD 3000 2200 3400 2SQO 3300

(b)

(c)

F igu re 5.7 C om parison o f mass spectra o f methanol crude extracts o f (a) heated pine (b) larch and (c)

rhododendron.

94

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5.4 Discussion

The research described in this chapter was aimed at testing the hypothesis that heating

woodchips o f pine (Pinus sylvestris), larch {Larix kaempferi) and rhododendron

{Rhododendron ponticum) would make them inhibitory to the pathogen P. ramorum.

Starting with micro-cosm studies in the laboratory, all three types of heat-treated

woodchips significantly inhibited the recovery of zoospores o f P. ramorum compared

with air-dried woodchips of the same species. Results of leaf disk assays o f the

inoculated woodchips also showed that not only zoospores of P. ramorum were able

to survive on air-dried wood chips, they were also able to give rise to sporangia with

all three species tested. A previous study on survival of P. ramorum in air-dried

firewood of tanoak has suggested that spores o f P. ramorum are able to survive up to

6 months in firewood under wet conditions. Twenty pieces of firewood (exposed to

natural inoculum and rainfall) were tested for the presence of viable zoospores as well

as chlamydospores using a similar baiting method to that used in this study (Shelly et

a l, 2006). Although, the rate of positive samples was low (14 out of 80), the results

suggested a 12% chance of the pathogen spreading from a piece of firewood to a

susceptible host under favourable conditions. The results o f the present study also

indicate that air-dried wood materials (in this case pine, larch and rhododendron) may

be able to support P. ramorum sporulation on the forest floor under condition o f high

inoculum levels. In contrast, when the same materials were heat-treated, they no

longer supported growth and zoospore production. Thus heating processed wood

after harvesting infected trees could be an effective method of reducing the risk of

pathogen establishment and spread when prolonged storage is needed. Furthermore,

since woodchips can be used as physical barriers to reduce the spread o f pathogens

such as P. ramorum, the use o f woodchips should be limited to species that do not

support survival and production of P. ramorum when used against this pathogen.

In the next step of the study, the extent to which the inhibitory effect might be altered

by mixing with soil was assessed. As all three heat-treated types of woodchip showed

similar inhibitory effects, only larch was tested in a mixture with soil. Heated larch

woodchips completely inhibited the recovery o f zoospores mixed in a 75:25% ratio

with soil, whereas when perlite was substituted for the woodchips, P. ramorum was

frequently recovered. These results are important since P. ramorum is known to be

able to survive and even produce sporangia in the litter interface with soil and foliage

96

(Fichtner et a l, 2007a). Survival and sporulation of P. ramorum in foliage is an

important stage in the pathogen life cycle. It allows spore inoculum to build up in

sufficient quantities to cause infection o f mature trees when temperature and moisture

conditions are favourable. Therefore, it is crucial to control pathogen populations

through control measures which reduce inoculum levels. The results o f using heated

larch woodchips mixed with soil also showed their inhibitory activity is retained

under moist conditions. This suggests the heat-treated woodchips could have the

potential to control pathogen survival under wet conditions, and even when mixed or

in contact with soil interface. However, the inhibitory effect may vary under natural

soil moisture conditions and needs to be investigated further.

Similarly, mixtures of heated wood of larch with soil (75:25) were inhibitory to

chlamydospores o f P. ramorum. After 4 weeks in the larch/soil mix only 5% of

recovered leaf disks containing chlamydospres supported outgrowth o f the pathogen,

compared with 90% recovery with perlite/soil treatments and 100% recovery with soil

only. Results were consistent even after 12 weeks of incubation. Lack o f recovery of

P. ramorum from pre-colonized rhododendron baits placed in different potting media

has been also reported (Linderman and Davis, 2006). Insufficient moisture and

presence o f inhibitors in the substrate are considered to be the major factors causing

this. This study suggests that the inhibitory effect o f heated wood o f larch could give

long term suppression o f the germination o f chlamydospores at least in a moisture

controlled environment. However, the long term effect could reduce under natural

conditions, as a result of rainfall leaching the inhibitory compounds from the

woodchips.

In order to identify potential inhibitory components of heated wood, crude extracts

were initially tested on zoospores, and later tested on myeelia and chlamydospores.

The preliminary tests showed the responses o f two P. ramorum isolates to crude

extracts are dose dependent. Although, all three crude extracts of heat-treated wood

species had an inhibitory effect on P. ramorum zoospores, pine and larch had a

greater effect than rhododendron (6.25 mg ml" ̂ vs. 50 mg ml'^). Phytophthora

ramorum isolate BRCOl also showed more tolerance in comparison with isolate

P I578. Unexpectedly, air-dried pine and larch also had inhibitory effect on isolate

PI 578 while none of air-dried crude extracts had any effect on BRCOl.

97

The same extracts significantly reduced germination of chlamydospores by more than

90% compared with extracts from air-dried wood. This may be inhibition rather than

lethal effect of the extracts. Since all three tested wood species showed similar

inhibitory effects, the potential inhibitors are likely to be produced through chemical

modification in structural components of wood upon heating. Chemical analysis of

the three crude extracts showed some o f the induced compounds are present in the all

three crude extracts but in different concentrations. Spectra of the three crude extracts

had vanillin, vanillic acid and syringaldéhyde, but the peak for vanillin was

considerably higher in the extracts of pine and larch compared to rhododendron.

Interestingly, coniferaldehyde was only present in larch and pine extracts, whereas,

benzoic acid, syringic acid and sinapaldehyde were only present in the extract of

rhododendron. In general, pine and larch extracts contained the same compounds in

similar concentrations.

To determine precisely the active compounds of the extracts, purified compounds

dominant in the crude extract of heated pine including vanillin, aeetovanillone and

coniferaldehyde were tested on zoospores, chlamydospores and mycelium o f the two

isolates of P. ramorum. Pure compounds were 2-10 times more active against all three

propagules types compared with the crude extracts. Coniferaldehyde was the most

active compound against myeelia, chlamydospores and zoospores o f P. ramorum.

Since coniferaldehyde was not found in the crude extract o f heat-treated

rhododendron, this may explain why pine and larch extracts had much a greater effect

compared to rhododendron. Aeetovanillone and vanillin had similar antimicrobial

properties against myeelia, chlamydospores and zoospores. Both coniferaldehyde and

vanillin have previously been found to exhibit antifungal activity against the wood

decay fungi Trametes versicolor and Coniphoraputeana (Voda et a l, 2003). Garbber

et al., (1998) conducted a cell-wall model system to evaluate what effect aldehyde-

containing lignins have on the hydrolysis o f maize cell walls by fungal enzymes

(Grabber et a l, 1998) and their finding revealed severe inhibition of wall degradation

occured with coniferaldehyde. As phenolic aldehydes, both compounds are known to

be the most toxic heartwood components of conifers. Also, a previous study by Nagle

et al (2011) suggests Quercus spp trees with field-resistance to P. ramorum contain

high level of phenolic components compared with susceptible trees. Overall it can be

concluded that high levels of phenolic aldehydes that are induced as a result o f

98

heating are likely to be responsible for the enhanced inhibitory effect of heat treated

woods. However, results of chemical analysis and the bioassays o f identified

compounds already presented in Chapter 4 suggest that the magnitude of

antimicrobial activity may not only be due to aldehydes since their effects are known

to be dose dependent. As Chapter 4 revealed, resin acids contribute a greater effect

when working in conjunction with aldehydes.

To test the hypothesis that resin acids contribute toward enhanced antimicrobial

activity, individual compounds were tested in combination with the resin acids

already identified in Chapter 4. Dehydroabietic acid, decreased the minimum

inhibitory effect o f all three tested phenolic compounds while it showed no effect

when tested separately against P. ramorum. Dehydroabietic acid, the dominant resin

acid present in heated crude extracts o f both pine and larch has shown antifungal

activity against Trametes versicolor and Trichoderma species (Savluchinske-Feio et

a l, 2007). The oxidized derivatives of dehydroabietic acid are reported to have good

antimicrobial activity against Dothistroma pini, a pathogen of Pinus species and

Heterobasidion annosum, a root pathogen of conifers (Franich et a l, 1983; Henriks et

a l, 1979; Savluchinske-Feio et a l, 1999). When Savluchinske Feio et a l, (2007)

studied the potential o f synthetic derivatives of dehydroabietic acid as a conventional

fungicides, they found it inhibited normal growth of Botrytis cinerea and

Lophodermium seditiosum. Dehydroabietic acid derivatives exhibit activity against a

wide range of bacteria and fungi (Savluchinske-Feio, et a l, 1999). These derivatives

may be potential replacement for wood preservatives with less environmental impact

(Savluchinske-Feio et a l , 2007).

Interestingly, abietic acid showed similar results with just one isolate of P. ramorum

(BRCOl) while pimaric acid had no effect in combination with any of the tested

compounds. The tested resin acids are diterpenoids which are found in oleoresin

produced by conifers. They are known to have low toxicity toward fungal pathogens

but are of importance in sealing wounds and trapping insect invaders (Phillips and

Croteau, 1999). Abietic acid and its derivatives have also been found to show

antifungal activity against Fusarium moniliforme and Aphanomyces euteiches

(Spessard et a l, 1995). Moreover, there is evidence that resins are constitutively

produced and stored in conifers and are part o f their defence mechanisms towards

insect and fungi invaders (Lewinsohn et al., 1991). In some conifers, resin ducts are

99

produced as an inducible defence mechanism, including western red cedar in response

to Armillaria ostoyae or European larch in response to physical injury (Bollschweiler

et a l, 2007; Cleary, 2010).

Based on this theory, the red discoloured bark o f Japanese larch suspected o f being a

defence reaction toward P. ramorum, was chemically analysed to provide evidence o f

compounds induced during pathogen invasion. The analysis o f the DCM crude

extracts revealed that prior to detection o f the pathogen in invaded bark, white phloem

tissue has an abundance o f resin acid 2 (dehydroabietic acid) and much lower

amounts o f resin acid 1 (methyl abietate, an AB derivative) compared with the

phloem from the healthy tissue (A 2 o,2 i)- However, in the crude extract o f the red-

discoloured tissue where the pathogen could not be detected, methyl abietate and

DHAA were both present in considerable quantities, whereas in the brown tissue

where detection o f the pathogen was successful, DHAA had reduced to levels similar

to the control. It was also striking that particularly the red discoloured tissue had

raised levels o f many other compounds when compared with healthy and white

coloured phloem tissue. One other noticeable change in the chemical components o f

discoloured bark was the presence o f a-pinene and 3-carene in the extract o f red and

dark brown phloem (A2 2 ,2 3 ). A similar phenomenon was reported after artificial

inoculation o f pine seedlings ^vith six different strains o f fungi which resulted in

higher biosynthesis o f volatile compounds especially a-pinene and 3-carene

(Napierala Filipiak et a l, 2002). Both compounds are monoterpenes found in the

essential oils o f conifers that exhibit toxic activity against fimgi and insects (Phillips

and Croteau, 1999). These results suggest that resin acids may play an important role

in inhibiting the growth o f P. ramorum after invasion but the overall inhibitory effect

could potentially be due to the presence o f other antimicrobial compounds that are

induced in small quantities in conjunction with higher concentrations o f resin acids.

Results o f the first field trial using heat-treated and air-dried woodchips were

consistent with the bioassay results o f crude extracts. A t fifty-two weeks after the

placement o f the bags o f woodchips on site, there was a more than two-fold increase

in recovery o f P. ramorum from air-dried larch and pine compared with heat-treated

samples. In contrast, P. ramorum was isolated from both heat treated and air-dried

rhododendron wood chips after 52 weeks, but at lower levels from the heat treated

woodchips although this was not significantly different from the air-dried woodchips.

100

The outcome can be explained by the results of the chemical analysis that revealed the

lack of coniferaldehyde in rhododendron wood which had the greatest inhibitory

effect in bioassays of isolated compounds. The crude extract of heat treated wood of

rhododendron also contained a lower concentration of vanillin in eomparison with

larch and rhododendron. Another major factor could be the lack o f diterpenoid resin

acids that naturally exist in conifers and were shown to considerably decrease the

MIC of active compounds. An inhibitory effect of the heated crude extract of

rhododendron was achieved at a high concentration of 50 mg ml'^ in bioassay tests, so

it can be concluded this was due to additive effect of the complex mixture of

compounds found in the crude extract. These are probably less effective under high

inoculums level of P. ramorum which can occur at certain time of year under natural

conditions. The lack o f differences between air-dried and heated woodchips after 4

and 1 2 weeks after natural exposure could also be explained by low levels of

inoculum at that time of sampling during the field trail.

In the second field trial, traditional baiting combined with a PCR methods using m-

RNA to detect live cells of P. ramorum was used to improve detection. However, the

field trial still failed to provide information about the inhibitory effect o f heated

materials apparently because too little natural inoculum was generated during the trial.

To determine the minimum detection limit of the RT-PCR assay, a dilution series

between 1 to 1,000,000 zoospores ml'^ in sterile water was tested. The minimum

detection limit was 10"̂ zoospore in 1 ml of water for the RT-PCR assay, suggesting

that the amount o f inoculum sampled from the field exposed woodchips was often

below the detection limit of tested PCR assay. Although it seems unlikely that the

negative results of the baiting method were also associated with a lack o f spores, an

alternative method of detecting inoculums could improve the chance o f comparing

treatments more effectively.

Extensive research on the management of P. ramorum indicates that there is no single

strategy to eradicate the pathogen once it has already established in the wider

environment. The best management praetices are focused on slowing the spread o f the

disease particularly in woodland and forest, but bio-security and quarantine efforts are

more difficult to implement successfully in these environments. As the spores are the

key factor in pathogen survival and spread, methods which can reduce sporulation and

dispersal are potentially the most effective. One strategy that has, been suggested is to

101

cover infected leaf litter on the floor o f a contaminated area by a layer o f woodchips

to reduce the movement of inoculum and prevent spread to nearby susceptible plants

(Goheen et a l, 2012; Manter et a l, 2007). This has potential to be effective in places

where human activities are concentrated, such as roads, walk ways in woodlands and

paths in parks and gardens. Moreover, use of woodchips material with antimicrobial

properties could give added protection if used as a part of disease management

practices. A research study by Manter et a l, (2007) evaluating antimicrobial activity

of yellow cedar against P. ramorum showed consistency o f bioassay results of

chemical components compared with woodchips used in field treatments. Similarly,

results of the present study showed heat-treated wood of larch and pine exhibit

inhibitory effect against the pathogen under natural conditions as well as laboratory

experiments. These results suggest that using heat treated materials has the potential

to be an effective method to reduce survival of the pathogen under natural conditions.

Moreover, the active chemicals of heated material can be the potential candidates for

further studies in the development of chemicals treatment for sudden oak disease.

102

C H APTER 6: DISCUSSIO N

Despite the wide application of heating techniques to improve the way that wood

materials are used in sectors such as construction, wood preservation and food

paekaging, a review of the literature revealed that there has been little attention to the

use of heat treatment in other areas such as animal bedding or disease control and

management in agriculture or forestry. Therefore, the studies reported here aimed to

investigate the potential of a heating technique to enhance the antimicrobial activity of

wood materials with potential application in two different fields: animal husbandry

and forestry.

The first objective of this study was to optimise a heating regime for pine wood

shaving materials. A preliminary study indicated that pre-heated wood shavings can

inhibit an important food-borne pathogen of chickens. Salmonella Enteritidis. The

second objective was to investigate and evaluate the effect o f enhanced inhibitory

activity on ammonia emission of heated wood materials in poultry bedding. The

investigation of inhibitory activity was performed by extraction of heat-treated and

air-dried wood materials using organic solvents and water. Chemical analysis

techniques (GC-MS and NMR spectroscopy) were employed to study the chemical

modification o f heat-treated pine wood shavings and its association with any new

inhibitory effect in a bio-active methanol extract. The dominant chemicals derived

from fractionation o f methanol extract were tested for their antimicrobial activity on

Salmonella Enteritidis, but the wider spectrum of antimicrobial activity was also

tested against other poultry bedding microorganisms including: Staphylococcus

aureus, E. coli and Saccharomyces cerevisiae. In the final part o f the study, the

application of heat-treated wood materials was extended to Phytophthora ramorum as

a potential control for this invasive pathogen of larch in UK forests. The study

consisted of applying the heating technique to wood materials o f Japanese larch and

rhododendron, the two main hosts of P. ramorum. The potential inhibitory effect o f

the heat-treated materials on P. ramorum was studied and tested by chemical analysis

techniques and bioassay development using some of the approaches developed for

poultry bedding and Salmonella Enteritidis.

To expand on the findings of a preliminary study which demonstrated that heated pine

wood shavings exhibit antimicrobial activity against Salmonella, temperatures

ranging from 20°C to 200°C was tested to evaluate and optimize the effect to enhance

103

antimicrobial activity. Interestingly, raising the temperature (from 50°C to 200°C)

and the length of time of heat treatment increased the inhibitory effect o f pine wood

against Salmonella Enteritidis. Further evaluation studies revealed a heating regime

o f 120°C for 72 hours reduced the number o f Salmonella recovered from inoculated

heated wood shavings and also reduced levels o f ammonia produced in the chicken

litter over at least 11 days. In addition, hardwoods such as beech and ash exhibited

antimicrobial activity against Salmonella Enteritidis as well as softwoods such as pine

and cypress, indicating the potential o f heat treatment to produce a range of suitable

poultry bedding materials in response to shortages in the market by introducing

antimicrobial properties across a wide range of wood species. However, this study

did not investigate the efficacy o f heated wood materials in the natural environment of

a broilers house, but it is worth noting that the heating technique also reduced the pH

of pine wood. This process could be used to produce bedding which is a much less

favourable environment for Salmonella.

In investigating the potential of using similar heating techniques to produce

antimicrobial woodchips against P. ramorum, heated wood of two susceptible hosts,

larch and rhododendron was found to inhibit P. ramorum zoospores when in direct

contact. Therefore, heat-treated larch wood showed promise for further evaluation in

the laboratory experiments. Heat-treated larch wood in mixtures with soil was also

effective at inhibiting P. ramorum zoospores and chlamydospores, indicating the

potential of the inhibitory effect can be extended to an ecologically more complex

environment. The field evaluation (Largin Wood, Cornwall) using pine and larch

woodchips indicated that the inhibitory activity brought about by heating could be

effective in reducing the recovery of P. ramorum from retrieved wood samples left in

the field for several weeks. In comparison, rhododendron woodchips were not as

effective as larch or pine even after heat treatment. Overall, the results suggested that

pressure of high inoculum in the field may affect the effectiveness of inhibition for

different wood species, and this outcome was later found to be associated with

differences in the chemical content of each wood species.

In identifying the chemical components responsible for the antimicrobial activity in

pine, larch and rhododendron, heated wood materials were extracted and the methanol

crude extracts showed antimicrobial activity in in vitro assays. Antimicrobial activity

was associated with the presence of aromatic compounds and an increase in more

104

saturated types of resin acid in the heat-treated wood. Chemical analysis of the active

methanol crude extract of heat treated wood of pine showed that it contains three

different groups of chemicals; a) aromatic compounds including benzaldehyde,

coniferaldehyde, aeetovanillone, vanillin and stilbene; b) organic acids including

formic acid, acetic acid, and vanillic acid; c) diterpenoid resin acids including

pimaric, abietic and dehydroabietic acid. Vanillin and coniferaldehyde were the

dominant compounds of the methanol crude extracts of pine and larch and are known

to be formed as a result o f lignin breakdown. They also occur naturally in essential

oils, herbs and spices and have previously been reported to inhibit S. Enteritidis, E.

coli, and S. aureus (Seydim and Sarikus, 2006). Extracted from vanilla beans,

vanillin exhibits several bioactive properties including antimicrobial activity against

yeasts, moulds and bacteria including Salmonella Enteritidis (Fitzgerald, 2004;

Mourtzinos et ah, 2009). Coniferaldehyde gives whisky and wine their fruity flavour

and has antioxidant and anticancer properties (Connors et a l, 1970). Both

coniferaldehyde and vanillin have previously been found to exhibit antifungal activity

against the wood decay fungi Trametes versicolor and Coniphora puteana (Voda et

a l, 2003). The overall results of bioassay tests of the fractionated compounds and

mixtures derived from the methanol crude extract in the present study demonstrated

however, that the enhanced antimicrobial effect was not usually due to one individual

compound. Fractions containing aromatic compounds and resin acids including

dehydroabietic acid and abietic acid showed a stronger effect on the tested pathogens

than when used singly. For example vanillin and stillbene were less effective when

they were tested individually on Salmonella Enteritidis and Staphylococcus aureus,

respectively. Similarly when investigating the role o f chemical components of heat-

treated larch and rhododendron and their inhibitory properties on P. ramorum,

coniferaldehyde, vanillin, aeetovanillone and resin acids including abietic and

dehydroabietic acid, were found to work best in combination. Dehydroabietic acid

was identified as the dominant compound present in active fractions containing

vanillin and stillbene. It is interesting that the resin acids dehydroabietic acid or

abietic acid had no inhibitory effect on Salmonella or P. ramorum when tested alone,

suggesting that mixtures of the active compounds and resin acids synergize to produce

a greater level of activity against both pathogens. An extensive literature review did

not reveal any similar findings so this is the first report of synergistic effect between

aromatic compounds and dehydroabietic or abietic acid.

105

These findings appear to be further supported by the results of chemical analysis of

discoloured larch bark in the present study. This showed that the increase in resin acid

content of bark is followed by the production of new compounds, a-pinene and 3-

carene, in the extract o f red and dark brown phloem which are formed after invasion

by P. ramorum. Both compounds are monoterpenes found in essential oils of conifers

that exhibit toxic activity against fungi and insects (Phillips and Croteau, 1999).

Furthermore, in some conifers, resin ducts are produced as an inducible defence

meehanism, such as in western red cedar in response to Armillaria ostoyae infection

(Cleary, 2010). Also higher concentrations of resin acids have been reported in Scots

pine heartwood which is decay-resistant to brown-rot, with abietic acid being the

most abundant (Harju et a l, 2005). It is notable that the complex composition of

oleoresin, which is known to be an important part of their successful defence

mechanism of conifer species against wide range of pests and pathogens, contains

both resin acids and monoterpenes (Lewinsohn et a l, 1991). The composition of

oleoresin from conifers has also been shown to contain significant amounts o f resin

acids (60-70%).

The results of bioassay tests of the chemical constituents o f heat treated wood on P.

ramorum may also suggest that the aromatic content o f different wood species

(particularly phenols) work in conjunction with resin acids to induce resistance

against different pathogens. A previous study conducted by Nagle et al. (2011)

showed that oak trees with field-resistance to P. ramorum contained high levels of

phenolic components compared with susceptible trees. In contrast, the inner bark of

conifers with higher phenolic content can be more susceptible to pathogen

colonization and wood decay (Kelsey and Harmon, 1989). This may indicate that the

phenolic content of conifers is not the only chemical constituent o f their defence

mechanisms. A research study by Harris and Webber (unpublished work) on the

susceptibility of larch to P. ramorum examined colonization o f Japanese, hybrid and

European larch by the pathogen, and found that at certain times of year, Japanese and

hybrid larch were more resistant than European larch. Research on the chemieal

constituents of the three species of larch indicates that Japanese and hybrid larch both

contain more resin acids, mainly abietic acid, compared with European larch. There

is evidence that resins are constitutively produced and stored in conifers and are part

o f their defence mechanisms toward insect and fungi invaders (Lewinsohn et a l.

106

1991). Abietic acid is known to have low toxicity toward fungal pathogens but is of

great importance in sealing wounds and trapping insect invaders (Phillips and

Croteau, 1999). It has also been shown to have phytoalexin properties which appear

to have an important role in inducing resistance in plant hosts. In addition,

dehydroabietic acid, the dominant resin acid present in heated crude extract of pine

and larch, has shown antifungal activity against Trametes versicolor and Trichoderma

species. Therefore, presence of these resin acids may explain the difficulty of

isolating P. ramorum from bark of infected larch species. However, unexpectedly,

Japanese larch has proved more susceptible to P. ramorum in the natural environment

in the UK, despite higher levels o f resin acids. This may be because infected foliage

of Japanese larch produces large numbers of spores and counteracts the inhibitory

effectors o f the resins acids. Alternatively, it could be explained by insufficient

concentrations o f resin acids produced by Japanese larch growing under UK

environmental conditions. To produce resin acids, conifer species need adequate

sunlight and this or other environmental conditions may play a significant part in tree

susceptibility in ways that are not yet fully understood.

Although, the mechanism of action of bio-active resin acids is not well understood,

research studies hypothesize that their antibacterial and antifungal properties result

from their interaction with the cell membrane of the target microorganism (Urzua et

a l, 2008) (Figure 6.1). The lipophilicity of these chemicals is thought to be

associated with their relative activity in the disruption of cell membranes

(Savluchinske-Feio et a l, 2006). Higher lipophilicity means a higher concentration of

the chemicals are able to bind with the cell membrane layer, resulting in stronger

activity. Resin acids with a high lipophilicity are used as the starting material in the

development of surfactants (Piispanen et a l, 2002). These chemicals act as soluble

surfactants in alkaline conditions, but in acidic conditions they only dissociate partly

depending on their pK values (Werker and Hall, 1997). They also are less lipophilic

and more water soluble under acidic conditions. This suggests that the mechanism by

which dehydroabietic acid/aldehyde showed greater antimicrobial activity in this

study was not due to dehydroabietic acid interfering with cell membrane function.

Instead, because the effect of temperature on heat-treated wood showed the pH of

pine could reduce to a value of 3.5 after 72 hours, the effect could be due to the acidic

107

conditions. On the other hand, vanillin showed antimicrobial activity against

Salmonella, but it was dose dependent and the activity was lost by dilution.

W (Hin€llllg

POG

0 0

/ \

Figure 6.1 Interactions o f secondary m etabolites w ith biom em brane and m em brane proteins.1, Sarsaparilloside (a steroidal saponine), 2, ocim ene (linear m onoterpene), 3, a -p inene (bicyclic m onoterpene), 4, cadinene (sesquiterpene), 5, phyllocladene (diterpene); (A) ion channel, (B) transporter, (C) m em brane receptor (W ink, 2006).

Currently, the mode of action of vanillin is unclear, although other phenolic aldehydes

act by disrupting cell membrane function (Fitzgerald et a l, 2004). Therefore, it can

be hypothesized that vanillin disrupts the cell membrane and allows dehydroabietic

acid to pass through cell membrane which may affect metabolic processes inside the

cell. The weak solubility of resin acids, which causes variability in the results of their

bioassay outcome, supports the idea that if disrupting membrane function is facilitated

by cell membrane active compounds such as vanillin, they should be more effective.

This also can be extended to the strong antimicrobial activity of coniferaldehyde/

DHHA mixture against P. ramorum. Grabber et al. (1998) demonstrated that severe

inhibition of wall degradation occurred with coniferaldehyde, using a cell-wall model

system to evaluate what effect aldehyde-containing lignins have on the hydrolysis of

maize cell walls by fungal enzymes (Grabber et a l, 1998). These findings are

important as they bring insight to the evaluation and application of resin acids and

their unique potential in development of antimicrobial pesticides and drug design.

108

The results o f the first field trial using heat-treated and air-dried woodchips on P.

ramorum were consistent with the bioassay results of crude extracts, providing further

evidence in the role of dominant aldehyde/resin acids on the inherent inhibitory

activity of heat-treated wood. The lack of coniferaldehyde which had the greatest

inhibitory effect in bioassays o f isolated compounds in heat-treated rhododendron

together with the lack o f diterpenoid resin acids (both major differences revealed by

chemical analysis) could explain the insignificant effect o f heat-treatment on

rhododendron in both laboratory experiments and also under field conditions. These

findings may indicate that all types of wood treated materials (softwood or hardwood)

can exhibit antimicrobial effects after heat-treatment because of the high content of

active aromatic compounds, but the level of activity may change under environmental

circumstances. Therefore, softwoods which contain diterpenoid resins could provide

stronger activity over a longer period.

In the latest review of wood waste carried out by DEFRA, in 2012, approximately 4.1

million tonnes of wood material was transferred into the waste stream in the UK

(Defra, 2012). O f this, the market for animal bedding materials from the UK total

wood machine waste usage was 56% which is estimated to be approximately 400,000

tons of recycled materials (Fletcher et a l, 2010). Results of this study suggest that,

heating wood shavings o f pine (I20°C for 72 hours) is potentially an effective

technique to produce antimicrobial wood materials for use as bedding in poultry units

and it could return larger amounts of wood waste into new uses. However, it is not

easy to estimate the efficiency level because o f the potential running costs of various

methods of heating. In addition, the efficiency of these materials still needs to be

tested for their effect in a real broiler environment for final evaluation. The benefits

gained from heat treatment of wood materials aimed as broiler bedding with improved

control of Salmonella and ammonia production could improve the life standards of

chickens and quality of poultry products.

Heat-treated wood of larch and pine was shown to exhibit an inhibitory effect against

P. ramorum under natural as well as under laboratory conditions. Use o f such

material in P. ramorum affected areas has the potential to be very effective in places

where human activities are concentrated such as roads, walk ways in woodlands, as

well as paths in parks and gardens. To prevent transfer of spores by people, use of

woodchip material with enhanced antimicrobial properties could give added

109

protection as a part o f disease management practices. Finally, the active chemicals of

heated material can yield potential candidates for further studies in the development

of chemicals for treatment of sudden oak death. It is also worth noting that large

quantities of wood waste are unrecyclable because of the bonding resins they contain.

(Fletcher et a l, 2010), so finding uses for this material would be economically and

environmentally useful.

Future work

The results of this study demonstrated that the heating technique made different wood

species antimicrobial against a number of organisms. Pine wood shavings showed

antimicrobial activity against Salmonella in both laboratory experiments and also

microcosom systems. Further studies are required to investigate the true value of

antimicrobial wood shavings under the natural environment o f broilers units. To be

able to evaluate the potential of antimicrobial wood shaving, trials using recycled pine

that is available in the market as poultry bedding should be set and run in parallel for

comparison. Also the true potential o f heated wood materials in reducing ammonia

production in poultry litter should be tested in a real broiler system. In this study

there was insufficient time for the potential o f other heated wood species to reduce

ammonia production to be investigated. It would be interesting to examine the wood

species that showed antimicrobial activity against Salmonella for their effect on

ammonia production.

Based on the results of this study, the pH of wood decreased to a value of 3.2 after the

heat treatment. The presence o f organic acids such as formic, acetic and vanillic acid

in the heated materials gives heated wood its acidic pH and may also be used to

advantage for controlling Salmonella in broilers units. Acetic acid, formic acid, lactic

and propionic acids are already commonly used to suppress Salmonella in animal

feeds (Martin and Maris, 2005). In addition, the low pH o f heated wood materials

may reduce the need for litter amendments to control pH of the litter. It may also help

to reduce the risk of emerging resistant pathogens of poultry which is believed to be

driven by exposure to excessive amount of chemicals used to control litter conditions.

One approach therefore might be to undertake comparative studies to evaluate which

of the heated wood species or acidifying amendments are more effective in

controlling litter pH.

110

Based on results of in vitro assays in this study, the magnitude of antimicrobial

activity was due to the presence of bio active diterpenoid resin acids operating in

conjunction with phenolic aldehydes in softwoods such as pine and larch. It would be

necessary to investigate the mechanism of this antimicrobial activity further. This

study hypothesized that the synergism between vanillin and dehydroabietic acid may

be explained by the role of vanillin in disrupting cell membrane and therefore

facilitating dehydroabietic acid transition through the cell membrane. A better

understanding of interactions between mixtures of bioactive compounds and their

target sites would provide important information on extending the application o f both

aldehydes and the resin acids. Further studies could also be extended to the

evaluation of antimicrobial activity o f other cell membrane active compounds in

combination with the resin acids. Using cell membrane active compounds could help

to reveal the potential of other antimicrobials which cannot actively pass through the

cell membrane. As such, there is no report on the antimicrobial activity of

dehydroabietic acid on Gram negatives, which in mixture with vanillin, exhibited

strong antimicrobial effects in the present study.

Finally, apart from antibacterial and antifungal activity, abietane and dehydroabietane

derivatives have been found to have antiviral and antitumor properties, although only

oxidative forms are found to have cytotoxicity. More o f their oxidative derivatives

are synthesized and investigated as part o f screening new bioactive compounds with

antiviral and anticancer properties (G onzalez et a l, 2010). Therefore, another

approach in taking advantage of their unique potential could be to investigate their

true value in combination with cell membrane active compounds. This also may

encourage further studies in the formulation o f bioactive compounds in the search for

new antimicrobials and anticancer drugs.

I l l

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Appendices

Spectrum 1.1: Mass spectrum of (+)-dehydroabietic acid (CDCI3) ...............................1

Spectrum 1.2: NMR spectrum of (+)-dehydroabietic acid (CDCI3) ......................... 2

Spectrum 1.3: NMR spectrum of (+)-dehydroabietic acid (CDCI3) ........................ 3

Spectrum 2: NMR spectrum of picealactone A (CDCI3) ............................................4

Spectrum 3.1: Mass spectrum of vanillin (CDGI3) ............................................................ 5

Spectrum 3.2: NMR spectrum of vanillin (CDCI3) ....................................................... 6

Spectrum 3.3: NMR spectrum of vanillin (CDCI3) .......................................................7

Spectrum 4.1: Mass spectrum of vanillic acid (CDCI3) .....................................................8

Spectrum 4.2 :^H NMR spectrum of vanillic acid (CDCI3) ............................................... 9

Spectrum 4.3: NMR spectrum of vanillic acid (CDCI3) .............................................10

Spectrum 5.1: Mass spectrum of acetovanillone (CDC13)............................................. 11

Spectrum 5.2: NMR spectrum of acetovanillone (CDCI3) ........................................ 12

Spectrum 6 : NMR spectrum of guaicol (CDCI3) ...................... 13

Spectrum 7.1: Mass spectrum of stilbene (CDCI3) ......................... 14

Spectrum 7.3: NMR spectrum o f 3, 3 ’-dimethoxy-4, 4’-dihydroxy-stilbene(CD3O D ).................................................................................................................................. 15

Spectrum 7.2: NMR spectrum of 3, 3 ’-dimethoxy-4, 4’-dihydroxy-stilbene(CD3O D )........................................................................................................ 16

Spectrum 8.1: Mass spectrum of coniferaldehyde (CDCI3) ........................................... 17

Spectrum 8.2: NMR spectrum of coniferaldehyde (CDCI3) ......................... 18

Spectra 9.1: Mass spectrum of methyl abietate (DCM)................................................20

Spectra 9.2: Mass spectrum of dehydroabietic acid (DCM)........................................ 21

Spectra 9.3: Mass spectrum of a-pinene (D CM )............................... 22

Spectra 9.4: Mass spectrum of 3-carene (DCM)............................................................. 23

128

Scan 3738 (24.476 min): PINE.D\data.ms 23$.1

Abundance

285.185000

80000!

75000

70000

65000

60000

55000!

50000'

45000

40000

35000

197.130000

300.125000!

141.0

20000

15000 155.0117.091.0

10000 55.0 183.1169.0

69.15000

257.1

50 60 70 80 90 1 0 0 1 1 0 1 2 0 1 3 0 1 4 0 1 5 0 1 6 0 1 7 0 1 8 0 1 9 0 2 0 0 2 1 0 2 2 0 230240 250 260 270280 290 300311m /z->

Spectrum 1.1: Mass spectrum o f (+)-dehydroabietic acid (CDCI3)

1

Ilfij] c

u8es

a«£%

0

1 aiSZX

ClC/3

[|9J] 31 OL

OiZQ'Si-^ 99W81 — tZLÎ'iZ —t z % v n ^IOK'93-'9ei3'oe-v0089'££ 3Z06‘I 8301'.. ,

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)'8 8 - _Î-9 8 #L18i

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Ui6'9fHA|-'"

6880 881 W —

J L3 0

J I I L

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aiSzu

CLc/2

901. 8 tr Z 0z\

uûu

cou«S•I<4-0

1s-aiszX< s

I

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Abundance Average of 13.701 to 13.730 min.: MS Vanilin.D\data.msis;.o

900000

800000

700000

600000

500000

400000

300000

200000

100000

000000

900000

800000

700000

600000

500000

81.0400000

109.0300000

20000053.0

100000

252.9168.1 190.9207.0222. 280.9100 120 140 160 180 200 220 240 260 280 300

Spectrum 3.1: Mass spectrum o f vanillin (CDCI3)

5

uÛu

&R>0

1&

sX

<s

:

[|9J] 02 St 01.

W3K-9S-

t608'9Z-., iZZZ'LL-r SSS9 Z2 J

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swrzzitzoioci

ZESEZtl

ZZ06191

a

_ g

UQ

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T r[|0J]

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AbundanceI

600000j

580000!

560000j

540000Î

520000|

500000{

4800001

460000

440000

420000

400000

380000

360000

340000

320000

300000

280000

260000

240000

220000

200000

180000

160000

140000

120000

100000

80000

60000

40000

20000

0

Average of 15.824 to 15.938 min.: M6 Vanilic acid D\data.ms168.0

5 1 0

9 7 0

77

1250

148 8 206.9225.9 281.0300.1 3 8 1 0 355.11WSU ' T ' ! V T ' T T I— I T T - r r - ^ 1 ■ r-T -r -T - i- j - r T - n - j - i — r r - | - r f - ! ~ r j r - i ' f r - f , , , ' | .

m /z-> 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340 36C

Spectrum 4.1: M ass spectrum o f vanillic acid (CDCI3)

R

J]

EQ.a

q4"

in4"

oin

qin

uQU

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5400000

5200000

5000000

4800000

4600000

4400000

4200000

4000000

3800000

3600000

3400000

3200000

3000000

2800000

2600000

2400000

: 2200000 :

2000000

1800000

1600000

1400000123.0

1200000

1000000

800000

600000

400000

200000326.9 354.92 5 2 9 2 8 1 0178.9 206.9

40 60 80 100 120 140 160 180 ,2qq_ 2 2 p _ 2 4 q 2 6 q _ 2 8 p _ 3 p g _ 320 340_ 360m/z-->

Spectrum 5.1: Mass spectrum o f acetovanillone (CDC13)

11

uQ

O)cs'a

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1CL

ZXfSiX£3

CLüC

[|aj] ZL 01

__

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Abundance

8000000

7500000

7000000

6500000

6000000

5500000

5000000

4500000

4000000

3500000

3000000

2 500000

2000000

1500000

1000000

500000

0

51.1

89.0

180

152.1

115.0

Scan 2528 (17 .552 min): M4 stilbene.DXdata.ms

207.0 253 .0 281.0 327 .0

m /z -> 40 60 80 100 120 140 160 180 200 220 240 260 280 300 320 340

Spectrum 7.1 : Mass spectrum o f stilbene (CDCI3)

14

- (\

- m

- to

- »

aoQu

213. ty

01E

rorn«4-o

D-oo

[|9J] Zl 01

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Abundance

90000

80000

75000

70000

65000

60000

55000

50000

45000

40000

35000

30000

25000

20000

15000

10000

5000

51.0

Average of 17.752 to 17.775 min.: M7 CFA .DXdata.m;178.0

135.0

77.0107.0

161.0

207.0

252.9 28 1 .1

m /z-> 40 60 80 100 120 140 160 180 200 220 240 260 280 300

Spectrum 8.1: Mass spectrum o f coniferaldehyde (CDCI3)

17

oÛs«■o

«■a

sou010)a .

ZX

rioc

CLOO

[|3J] 21. 01.

S2W9S V 9t'S2’9S "

6090"221, mZ'ZiT 9899/2̂

0622 601-

2 2 kZ'S^-

9W9'Wt - 9209 92 0698 921^

UQU4̂%«■o

cou(4-o

BZVZ'IV̂ -iLn%n-OlfVÎSi-

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2 tWE6 l -

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“I— I— I— r[|SJ] 09

1 I r Ofr

1—I—I—r09 02

1 I I I r

01

> \ f c > u r » c l a r i œ

Unknown: Scan 3655 (24,001 min): brW"G1’15-fib-da.D\data.«ns Compound In Library Factor = -235

143100-

121

207

15550-

171193

0ISO 210120 150

256

241

275

316

301

394

210 240 270 300 330 360 390

Hit 1 : Methyl abietateC21H3202; MF; 7S2; RMF; 8Û7; Pmb 59.5%; CAS: 127-25-3; Lib: repiib; ID: 28885.

1 0 0

121105

21350- 131 185143

157171 I 195

04-180 210150120

256

241

273

4' I 270

31.

O301-)l', îjl̂ I ; I I’.-30Ô 330 360 390

Spectra 9.1 : Mass spectrum o f m ethyl abietate (DCM)

20

Unknown: Scan 3803 (24.847 min): h-0l-15-ati-cta,0\data,iins Compound In Library Factor = 640

100 -

50-

197

141

55 81 , j l I I, J i l l f 1 211225

285

I 300

Jii. ̂Ji.̂ 39660 90 120 150 ISO 210 240 270 300 330 360 390

Hit 1 : Detrydroabtedc addC20H2SO2; MF: 924; RMF: 968; Prob 63.6%; CAS; 1740-19-8; Lib: matnlib; 10:194581.

100-

50-

197

55 69 B'iVJ . .,1. 1,1,.. ?...

^^^55 Î IT169183M l 'lib -i

239I

257271211225

60 90 120 150 180 210 240 270 300 330 360 390

285

V V, . ^ x o r

300

z n I 7-^VV V"l ' < I I

OH

Spectra 9.2: Mass spectrum o f dehydroabietic acid (DCM)

2 1

/ k b u n d a n c

Unknown: Sean 527 {§.102 min): brw_<tam2.D\data.ms Compound In Library Factor = 101

100-

50-

83

77

53 .,1,1

60 60

sr

70 80

91

89■ • ’T90

> I ' 100

105 121

u s iig l^

Hitl : (iR)-2,6,§-Triïïî0thyiblcydoJ3.1.1ltifipt-2-eneC10H16; MF: 957; RMR 059: Pmb 25.2%; CAS: 7785-70-8; Lib: repiib; ID: 13150.

136

110 120 130 140 150

100-j

504

93

53, I

50

67

60 70

79

105 121

115119|.136

80 100 110 120 130 140 150

Spectra 9.3: Mass spectrum o f a-p inene (DCM )

22

/ A b u n d a n c e

Unknown: Scan 795 {7,578 min); brw_ {̂Jcm2.D\data,ms Compound in Library Facfor = -101

100-

50-

53

50 60

67u

77

93

60

01

| i89 89 i. .- |'i

90

105

. , .

100 110

115

121

119 = . 12 0

136

, I l-T-130 lîô" 150

Hit 2 : 3-CareneC1ÛH16; MF: 925; RMF; 926; Pmb 10.4%; CAS; 13466-76-0; Lib: æpiib; ID: 13141.

100

60-

77

5358

5772 75

I-- 5 • I ' I ' I M ' i I I - ;60 70

03

91

89r ' r " I "' I

80 90

121105

, i ' IMI -1 1 0 0

11511

136

110 120 1301 r - 1 - -I—<—T-i—140 150

Spectra 9.4: Mass spectrum o f 3-carene (DCM) 23

24